Bipolar trans carotenoid salts and their uses

ABSTRACT

The invention relates to trans carotenoid salt compounds, methods for making them, methods for solubilizing them and uses thereof. These compounds are useful in improving diffusivity of oxygen between red blood cells and body tissues in mammals including humans.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 10/372,717, filed Feb. 25, 2003, which claims thebenefit of U.S. Provisional Patent Application No. 60/358,718, filedFeb. 25, 2002, the entire contents of which are hereby incorporated byreference in this application.

FIELD OF THE INVENTION

[0002] The invention relates to bipolar trans carotenoid salt compounds,methods of solubilizing them, methods for making them, and methods ofusing them. These bipolar trans carotenoid salts (BTCS) compounds areuseful in improving diffusivity of oxygen between red blood cells andbody tissues in mammals including humans.

BACKGROUND OF THE INVENTION

[0003] Carotenoids are a class of hydrocarbons consisting of isoprenoidunits joined in such a manner that their arrangement is reversed at thecenter of the molecule. The backbone (skeleton) of the molecule consistsof conjugated carbon-carbon double and single bonds, and can also havependant groups. Although it was once thought that the skeleton of acarotenoid contained 40 carbons, it has been long recognized thatcarotenoids can also have carbon skeletons containing fewer than 40carbon atoms. The 4 single bonds that surround a carbon-carbon doublebond all lie in the same plane. If the pendant groups are on the sameside of the carbon-carbon double bond, the groups are designated as cis;if they are on opposite side of the carbon-carbon bond, they aredesignated as trans. Because of the large number of double bonds, thereare extensive possibilities for geometrical (cis/trans) isomerism ofcarotenoids, and isomerization occurs readily in solution. A recentseries of books is an excellent reference to many of the properties,etc. of carotenoids (“Carotenoids”, edited by G. Britton, S.Liaaen-Jensen and H. Pfander, Birkhauser Verlag, Basel, 1995 herebyincorporated by reference in its entirety).

[0004] Many carotenoids are nonpolar and, thus, are insoluble in water.These compounds are extremely hydrophobic which makes their formulationfor biological uses difficult because, in order to solubilize them, onemust use an organic solvent rather than an aqueous solvent. Othercarotenoids are monopolar, and have characteristics of surfactants (ahydrophobic portion and a hydrophilic polar group). As such, thesecompounds are attracted to the surface of an aqueous solution ratherthan dissolving in the bulk liquid. A few natural bipolar carotenoidcompounds exist, and these compounds contain a central hydrophobicportion as well as two polar groups, one on each end of the molecule. Ithas been reported (“Carotenoids”, Vol. 1A, p. 283) that carotenoidsulphates have “significant solubility in water of up to 0.4 mg/ml”.Other carotenoids that might be thought of as bipolar are also not verysoluble in water. These include dialdehydes and diketones. A di-pyridinesalt of crocetin has also been reported, but its solubility in water isless than 1 mg/ml at room temperature. Other examples of bipolarcarotenoids are crocetin and crocin (both found in the spice saffron).However, crocetin is only sparingly soluble in water. In fact, of all ofthe bipolar carotenoids, only crocin displays significant solubility inwater.

[0005] U.S. Pat. Nos. 4,176,179; 4,070,460; 4,046,880; 4,038,144;4,009,270; 3,975,519; 3,965,261; 3,853,933; and 3,788,468 relate tovarious uses of crocetin.

[0006] U.S. Pat. No. 5,107,030 relates to a method of making2,7-dimethyl-2,4,6-octatrienedial and derivatives thereof.

[0007] U.S. Pat. No. 6,060,511 relates to trans sodium crocetinate (TSC)and its uses. The TSC is made by reacting naturally occurring saffronwith sodium hydroxide followed by extractions.

[0008] In Roy et al, Shock 10, 213-7. (1998), hemorrhaged rats (55%blood volume) were given a bolus of trans sodium crocetinate (TSC) afterthe hemorrhage ended, followed by saline after another 30 minutes. Allof the TSC-treated animals lived, while all controls died. Whole-bodyoxygen consumption increased in the TSC group, reaching 75% of normalresting value after about 15 minutes.

[0009] Laidig et al, J Am Chem. Soc. 120, 9394-9395 (1998), relates tocomputational modeling of TSC. A simulated TSC molecule was “hydrated”by surrounding it with water molecules. The re-ordering of the water inthe vicinity of the TSC made it easier for oxygen molecules to diffusethrough the system. The computational increase in diffusivity of ˜30%was consistent with results obtained in both in vitro and animalexperiments.

[0010] In Singer et al, Crit Care Med 28, 1968-72. (2000), TSC improvedhemodynamic status and prolonged rat survival in a rat model of acutehypoxia. Hypoxia was induced using a low oxygen concentration (10%) airmixture: after 10 minutes the animals were given either saline or TSC.Hypoxemia led to a reduction in blood flow, and an increase in basedeficit. Only 2 of 6 animals survived in the control group. The treatedgroup all survived with good hemodynamic stability for over two hours,with a slow decline thereafter.

SUMMARY OF THE INVENTION

[0011] The subject invention relates to bipolar trans carotenoid salts(BTCS) compounds and synthesis of such compounds having the structure:

YZ-TCRO-ZY

[0012] where:

[0013] Y=a cation

[0014] Z=polar group which is associated with the cation, and

[0015] TCRO=trans carotenoid skeleton.

[0016] The subject invention also relates to individual BTCS compoundcompositions (including a TSC composition) wherein absorbency of thehighest peak (of an aqueous solution of the BTCS composition) whichoccurs in the visible wave length range divided by the absorbency of thepeak which occurs in the UV wave length range, is greater than 7.0,advantageously greater than 7.5, most advantageously greater than 8.

[0017] The invention also relates to a method of treating a variety ofdiseases comprising administering to a mammal in need of treatment atherapeutically effective amount of a compound having the formula:

YZ-TCRO-ZY

[0018] The invention also includes several methods of solubilizing andsynthesizing compounds having the formula:

YZ-TCRO-ZY

[0019] The invention also relates to an inhaler for delivery of thecompounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0020] A new class of carotenoid and carotenoid related compounds hasbeen discovered. These compounds are referred to as “bipolar transcarotenoid salts” (BTCS).

[0021] Compounds of the Invention

[0022] The subject invention relates to a class of compounds, bipolartrans carotenoid salts, that permit the hydrophobic carotenoid orcarotenoid related skeleton to dissolve in an aqueous solution, andmethods for making them. The cations of these salts can be a number ofspecies, but advantageously sodium or potassium (these are found in mostbiological systems). Commonly owned U.S. Pat. No. 6,060,511, herebyincorporated by reference in its entirety, describes an extractionmethod for making trans sodium crocetinate, TSC (a BTCS) starting fromsaffron.

[0023] A general structure for a bipolar trans carotenoid salt is:

YZ-TCRO-ZY

[0024] where:

[0025] Y (which can be the same or different at the two ends)=a cation,preferably Na⁺ or K⁺ or Li⁺. Y is advantageously a monovalent metal ion.Y can also be an organic cation, e. g., R₄N⁺, R₃S⁺, where R is H, orC_(n)H_(2n+1) where n is 1-10, advantageously 1-6. For example, R can bemethyl, ethyl, propyl or butyl.

[0026] Z (which can be the same or different at the two ends)=polargroup which is associated with the cation. Optionally including theterminal carbon on the carotenoid (or carotenoid related compound), thisgroup can be a carboxyl (COO⁻) group or a CO group. This group can alsobe a sulfate group (OSO₃ ⁻) or a monophosphate group (OPO₃ ⁻), (OP(OH)O₂⁻), a diphosphate group, triphosphate or combinations thereof.

[0027] TCRO=trans carotenoid or carotenoid related skeleton(advantageously less than 100 carbons) which is linear, has pendantgroups (defined below), and typically comprises “conjugated” oralternating carbon-carbon double and single bonds (in one embodiment,the TCRO is not fully conjugated as in a lycopene). The pendant groupsare typically methyl groups but can be other groups as discussed below.In an advantageous embodiment, the units of the skeleton are joined insuch a manner that their arrangement is reversed at the center of themolecule. The 4 single bonds that surround a carbon-carbon double bondall lie in the same plane. If the pendant groups are on the same side ofthe carbon-carbon double bond, the groups are designated as cis; if theyare on the opposite side of the carbon-carbon bond, they are designatedas trans. The compounds of the subject invention are trans. The cisisomer typically is a detriment—and results in the diffusivity not beingincreased. In one embodiment, a trans isomer can be utilized where theskeleton remains linear.

[0028] Examples of trans carotenoid or carotenoid related skeletons are:

[0029] where pendant groups X (which can be the same or different) arehydrogen (H) atoms, or a linear or branched group having 10 or lesscarbons, advantageously 4 or less, (optionally containing a halogen), ora halogen. Examples of X are a methyl group (CH₃), an ethyl group(C₂H₅), a halogen-containing alkyl group (C1-C10) such as CH₂Cl, or ahalogen such as Cl or Br. The pendant X groups can be the same ordifferent but the X groups utilized must maintain the skeleton aslinear.

[0030] Although many carotenoids exist in nature, carotenoid salts donot. Commonly owned U.S. Pat. No. 6,060,511 relates to trans sodiumcrocetinate (TSC). The TSC was made by reacting naturally occurringsaffron with sodium hydroxide followed by extractions that selectedprimarily for the trans isomer.

[0031] The presence of the cis and trans isomers of BTCS can bedetermined by looking at the ultraviolet-visible spectrum for thecarotenoid sample dissolved in an aqueous solution. Given the spectrum,the value of the absorbency of the highest peak which occurs in thevisible wave length range of 416 to 423 nm (the number depending on thesolvent used) is divided by the absorbency of the peak which occurs inthe UV wave length range of 250 to 256 nm, can be used to determine thepurity level of the trans isomer. When the BTCS is dissolved in water,the highest visible wave length range peak will be at about 421 nm andthe UV wave length range peak will be at about 254 nm. According to M.Craw and C. Lambert, Photochemistry and Photobiology, Vol. 38 (2),241-243 (1983) hereby incorporated by reference in its entirety, theresult of the calculation (in that case crocetin was analyzed) was 3.1,which increased to 6.6 after purification.

[0032] Performing the Craw and Lambert analysis, using a cuvettedesigned for UV and visible wave length ranges, on the trans sodiumcrocetin of commonly owned U.S. Pat. No. 6,060,511 (TSC made by reactingnaturally occurring saffron with sodium hydroxide followed byextractions which selected primarily for the trans isomer), the valueobtained averages about 6.8. Performing that test on the synthetic TSCof the subject invention, that ratio is greater than 7.0 (e.g. 7.0 to8.5), advantageously greater than 7.5 (e.g. 7.5-8.5), mostadvantageously greater than 8. For the TSC synthesized according to theimproved method of Example 5, the ratio is greater than 7.4 (e.g.7.4-8.5). The synthesized material is a “purer” or highly purified transisomer.

[0033] It has been found, recently, that TSC has an aqueous solubilityof around 10 mg/ml at room temperature, which is remarkable for amolecule containing such a long, hydrophobic portion. TSC has also beenfound to increase the diffusivity of oxygen through liquids.

[0034] U.S. Pat. No. 6,060,511 describes an extraction method for makingTSC starting from saffron; however, other bipolar carotenoid saltscannot be made using that same procedure since the use of saffron allowsonly a single carotenoid skeleton to be incorporated into the salt.

[0035] The invention disclosed herein allows the synthesis of a wholeclass of compounds: bipolar trans carotenoid salts which contain variouscarotenoid or carotenoid related skeletons. Such compounds are solublein aqueous solutions and have advantageous biological uses, such ascausing an increase in oxygen utilization. It is believed that thisincrease is a result of the ability of the hydrophobic portion (theskeleton) of the bipolar trans carotenoid salt to affect the bonding ofwater molecules. This, in effect, allows the oxygen molecule to diffusefaster in that area.

[0036] Solubilizing the Compounds and Compositions of the Invention

[0037] The invention allows for the dissolution of a trans carotenoid orcarotenoid related skeleton molecule in aqueous solutions. The novelmethods of dissolution are related below. The methods apply to anybipolar trans carotenoid salt and composition thereof.

[0038] BTCS-Containing Saline Infusion Solutions

[0039] Large volumes (as much as 3 times the estimated blood loss) ofisotonic saline (also called normal saline) are infused as a treatmentfor hemorrhagic shock. The isotonic saline contains 9 g NaCl per literof water so as not to disturb the ionic strength of the plasma once itis infused into the body. Adding TSC to the saline has been shown toresult in a superior infusion fluid, however, one cannot simply mix TSCpowder with the saline to make such a solution. About 50% of the TSCdissolves in normal saline no matter how much TSC is added (up toseveral milligrams per ml), which means that undissolved particles ofTSC are still present. In order to prevent that, a stock solution can bemade by adding more than twice the amount of TSC needed and thencentrifuging out the particles that do not dissolve. The actualcomposition of the stock solution can be verified using UV-visiblespectroscopy. This stock solution can be added to normal saline and theTSC remains dissolved.

[0040] This method can be used to dissolve a BTCS in other types ofsodium chloride solutions, as well as in solutions of other salts suchas KCl, Na₂SO₄, lactate, etc. Several, eg 1-3 mg/ml, can be put intosolution in this manner.

[0041] Dilute Solution of Sodium Carbonate Dissolves BTCS

[0042] A BTCS such as TSC dissolves in very dilute sodium carbonatesolutions. A dilute, eg 0.00001-0.001M, solution of sodium carbonate canbe added, dropwise, to deionized water until the pH is 8.0 (the pH ofdeionized water is usually 5-6). This only takes a few drops of the verydilute sodium carbonate per, say, 50 mls of deionized water. This sodiumcarbonate-deionized water solution is capable of completely dissolving alarge amount of TSC (around 10 mg/ml)—which is remarkable consideringthe hydrophobicity of the carotenoid portion of the BTCS.

[0043] A BTCS can be supplied as a powder along with a sterilized bottleof the sodium carbonate water. This concentrated solution can then beinjected directly (very small volumes of solutions having a lower ionicstrength than plasma can be injected), or the concentrated solution canbe added to normal saline and then injected. If TSC is dissolved in thesodium carbonate-water solvent and then more of the same solvent isadded—the TSC stays in solution.

[0044] In another embodiment, sodium bicarbonate is used instead ofsodium carbonate. Other salts which result in the deionized water havinga basic pH can also be used.

[0045] Carotenoid skeleton concentrations of 5-10 mg/ml can be achievedwith this procedure.

[0046] Water Dissolves BTCS

[0047] Although TSC dissolves in water (tap, distilled, deionized),these solutions are only stable if the pH is adjusted so as to make thesolution basic. TSC is more soluble in deionized water (very few Na⁺ions present) than in normal water. A BTCS, such as TSC, will dissolvein just deionized water alone, but, if plain deionized water is added tothat solution, the TSC will precipitate out. A BTCS will dissolve injust deionized water alone, but additional deionized water may causeprecipitation of the BTCS if the pH is not adjusted to make it slightlybasic.

[0048] Other Methods of Solubilizing BTSC

[0049] The BTCS can be formulated in a delivery system that enhancesdelivery. See Formulations of the Compounds of the Invention below.

[0050] Synthesis of the Compounds of the Invention

[0051] Bipolar Trans Carotenoid Salts

[0052] Set forth below are the novel synthesis methods that can be usedfor synthesizing bipolar trans carotenoid salts. There can be variationsin various steps of the synthesis that are obvious to one skilled in theart.

[0053] A. TSC Synthesis

[0054] Trans sodium crocetinate (TSC) can be synthesized by coupling asymmetrical C₁₀ dialdehyde containing conjugated carbon-carbon doublebonds (2,7-dimethylocta-2,4,6-triene-1,8-dial) with[3-carbomethoxy-2-buten-1-ylidene] triphenylphosphorane. This results inthe formation of a trans dimethyl ester of crocetin. This dimethyl esteris then converted to the final TSC product by saponification. Typically,saponification is accomplished by treating an ester with either aqueoussodium hydroxide or sodium hydroxide dissolved in THF (tetrahydrofuran);however, these methods did not give the best results in this case.Saponification can be accomplished very well, in this case, by reactingthe ester with an NaOH/methanol solution. After saponification, the TSCis recovered by drying in a vacuum.

[0055] The C₁₀ dialdehyde and the triphenylphosphorane reactants used inthis synthesis can be made via different routes. For example, the C₁₀dialdehyde was prepared starting with ethyl bromoacetate and furan usingWittig chemistry. Tiglic acid was the starting material for making thedesired phosphorane. Different lengths of carotenoid skeletons can bemade by joining together reactants of different lengths (for example aC₁₄ dialdehyde and triphenylphosphorane). This procedure results in theformation of different trans bipolar carotenoid salts. Alterations canalso be made so as to obtain different pendant groups (TSC has methylgroups for the pendant groups).

[0056] The TSC made in this manner is soluble in water (pH adjusted to8.0 with a very dilute solution of sodium carbonate) at a level >10mg/ml at room temperature. Other bipolar trans carotenoid salts aresoluble at room temperature in water having a pH that is neutral orabove. As used herein, “soluble” means that amounts greater than 5 mgwill dissolve per ml of water at room temperature (as noted previously,carotenoid references state that 0.4 mg/ml is “highly significantsolubility”—but that is lower than the subject definition ofsolubility).

[0057] B. General Synthesis

[0058] Carotenoid or carotenoid related structures can be built up inthe following manner:

[0059] (3-carbomethoxy-2-buten-1-ylidene)triphenylphosphorane (or arelated compound when X is other than a methyl group) is a key precursorto add isoprenoid units (or isoprenoid related units) to both ends of asymmetrical carotenoid (or carotenoid related compound). This processcan be repeated infinitely. For example, dimethyl trans crocetinate canbe reduced to the corresponding symmetrically dialdehyde using thechemistry described above. This dialdehyde can be reacted with excess(3-carbomethoxy-2-buten-1-ylidene)triphenylphosphorane to give thecorresponding diester. This synthetic sequence can be repeated again andagain.

[0060] Improved Synthesis

[0061] 2,7-Dimethyl-2,4,6-octatrienedial (2,7-dimethylocta2,4,6-triene-1,8 dial) is a key intermediate toward the synthesis ofTSC. This key precursor has three double bonds and thus several isomersare possible. For TSC, the all trans isomer (E,E,E-isomer) is required.The general synthesis route involves an 11-step synthesis withrelatively low yields and poor selectivity in several steps (see Example1). As a result, column chromatography is required to purify severalintermediates along the way.

[0062] The improved synthesis route is much simpler (see the reactionscheme below). The 3-step process as described in U.S. Pat. No.5,107,030, hereby incorporated by reference in its entirety, gives amixture of geometric isomers of the dialdehyde (U.S. Pat. No. 5,107,030does not note this mixture). In the method of the subject inventiondescribed in Example 1, 96-97% of the desired isomer (all trans orE,E,E-isomer) is obtained by several recrystallizations from methanol orethyl acetate in 59% yield.

[0063] The improved synthesis method of the subject invention involvesconverting the remaining isomeric mixture of dialdehydes into thedesired trans aldehyde (E,E,E) by isomerization with a sulfinic acid(RSO2H where R is C1 through C10 straight or branched alkyl group or anaryl group (a substituted phenyl group) such as para-toluenesulfinicacid, in an appropriate solvent such as 1,4-dioxane, tetrahydrofuran ordialkyl ether where the alkyl group is one or two of a C1 through C10straight or branched alkyl group. An additional 8% yield of the puredesired dialdehyde is obtained, raising the overall yield of the laststep from 59% to 67% yield. This yield improvement is important. Thisisomerization step can be incorporated into the third step of the methodof U.S. Pat. No. 5,107,030 to get a better yield.

[0064] Improved Synthesis Route:

[0065] Isomerization of Undesired to Desired Dialdehdye:

[0066] Saponification can be accomplished by dissolving the diester inmethanol and then adding a base such as NaOH (Y of the BTCS is thenNa⁺). Alternatively, the diester can be dissolved in methanol alreadycontaining the base. The NaOH is typically aqueous (20-60% by wt.) butcan be solid. Alternatives to methanol for dissolving the diester areethanol, propanol and isopropanol. Saponification can be carried out invarious ways commercially. A one or two phase system (one organic andone aqueous phase) can be used.

[0067] Trans crocetin can also be synthesized according to the methodsdescribed above.

[0068] In addition, as has been reported for TSC, such BTCS compoundsincrease the diffusivity of oxygen through water (this will also dependon the nature of the hydrophobic portion incorporated into the finalproduct such as carbon chain length) since it is believed that thehydrophobic interactions of the carotenoid skeleton with water result inthe increased diffusivity).

[0069] Formulations of the Compounds of the Invention

[0070] A concentrated solution of a bipolar trans carotenoid salt can bemade, as described previously, by dissolving it in a very dilutesolution of sodium carbonate. The resulting mixture can then be used inthat manner, or can be diluted further with normal saline or otheraqueous solvents. In addition, solutions of a bipolar trans carotenoidsalt can be made by dissolving the bipolar trans carotenoid saltdirectly in a salt solution and then getting rid of any material thatdoes not dissolve.

[0071] The bipolar trans carotenoid salts are stable in a dry form atroom temperature, and can be stored for long periods. Advantageously, aformulation of such salts, if given orally, is absorbed in the gut,rather than the stomach.

[0072] Although the compounds of the invention can be administeredalone, they can be administered as part of a pharmaceutical formulation.Such formulations can include pharmaceutically acceptable carriers knownto those skilled in the art as well as other therapeutic agents-seebelow. Advantageously, the formulation does not include a compound thatinhibits the ability of the compounds of the invention to improvediffusivity of oxygen.

[0073] Appropriate dosages of the compounds and compositions of theinvention will depend on the severity of the condition being treated.For a dose to be “therapeutically effective”, it must have the desiredeffect, i.e. increase the diffusivity of oxygen. This in turn, willcause oxygen-related parameters to return towards normal values.

[0074] Administration can be by any suitable route including oral,nasal, topical, parenteral (including subcutaneous, intramuscular,intravenous, intradermal and intraosseus), vaginal or rectal. Thepreferred route of administration will depend on the circumstances. Aninhalation route is advantageous for treatment in emergency situations,where it is necessary for the BTCS to enter the bloodstream veryquickly. The formulations thus include those suitable for administrationthrough such routes (liquid or powder to be nebulized). It will beappreciated that the preferred route may vary, for example, with thecondition and age of the patient. The formulations can conveniently bepresented in unit dosage form, e.g., tablets and sustained releasecapsules, and can be prepared and administered by methods known in theart of pharmacy. The formulation can be for immediate, or slow orcontrolled release of the BTCS. See for example, the controlled releaseformulation of WO 99/15150 hereby incorporated by reference itsentirety.

[0075] Formulations of the present invention suitable for oraladministration can be presented as discrete units such as pills,capsules, cachets or tablets, as powder or granules, or as a solution,suspension or emulsion. Formulations suitable for oral administrationfurther include lozenges, pastilles, and inhalation mists administeredin a suitable base or liquid carrier. Formulations for topicaladministration to the skin can be presented as ointments, creams, gels,and pastes comprising the active agent and a pharmaceutically acceptablecarrier or in a transdermal patch.

[0076] Formulations suitable for nasal administration wherein thecarrier is a solid include powders of a particular size that can beadministered by rapid inhalation through the nasal passage. Suitableformulations wherein the carrier is a liquid can be administered, forexample as a nasal spray or drops.

[0077] Formulations suitable for parenteral administration includeaqueous and non- aqueous sterile injection solutions that can containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient, andaqueous and nonaqueous sterile suspensions which can include suspendingagents and thickening agents. The formulations can be presented in unitor multi-dose containers, for example sealed ampules and vials, and canbe lyophilized, requiring only the addition of the sterile liquidcarrier such as water for injection immediately prior to use. Injectionsolutions and suspensions can be prepared from sterile powders, granulesand tablets.

[0078] Uses of the Compounds and Compositions of the Invention

[0079] A wide variety of conditions are controlled or are mediated bydelivery of oxygen to body tissues. The compounds and compositions ofthe subject invention can be used in the same pharmaceuticalapplications described for crocetin in the same effective amounts; seeU.S. Pat. Nos. 4,176,179; 4,070,460; 4,046,880; 4,038,144; 4,009,270;3,975,519; 3,965,261; 3,853,933; and 3,788,468 each of which is herebyincorporated by reference in its entirety.

[0080] TSC has been shown to increase the diffusivity of oxygen throughaqueous solutions by about 30%. TSC increases survival in mammalsfollowing hypoxia, increases oxygen consumption following hypoxia orphysiological stress, increases blood pressure following hypoxia,decreases blood acidosis (i.e., decreases blood base deficit, increasesblood pH, and decreases plasma lactate level) following hypoxia,decreases organ damage (e.g. liver, kidney) following hypoxia. Thus, thecompounds of the invention are useful for treating mammal (includinghuman) diseases/conditions which are characterized by low oxygen(hypoxia) such as respiratory diseases, hemorrhagic shock andcardiovascular diseases, multiple organ failure (due to, for example,ARDS sepsis or hemorrhagic shock), chronic renal failure,atherosclerosis, emphysema, asthma, hypertension, cerebral edema,papillomas, spinal cord injuries, stroke, among others. The compounds ofthe invention are also useful for treating mammals at risk for theabove-noted diseases/conditions. Other bipolar trans carotenoid saltshave similar properties. Such compounds can also be used in conjunctionwith other methods commonly suggested for increasing oxygen utilizationin the body, such as oxygen therapy and the use of hemoglobins orfluorocarbons.

[0081] In one embodiment of the invention, a BTCS is administered to thepatient while administering oxygen. Alternatively, hemoglobins orfluorocarbons and a BTSC can be given together. In these cases, anadditive effect is realized.

[0082] The minimum dosage needed for treatment for any of these salts isthat at which the diffusivity of oxygen increases. The effective dosageof the compounds of the inventions will depend upon the conditiontreated, the severity of the condition, the stage and individualcharacteristics of each mammalian patient addressed. Dosage will vary,however, from about 0.001 mg of active compound per kg of body weight upto about 500 mg per kg, and advantageously from about 0.01-30 mg/kg ofbody weight. IV administration is advantageous but other routes ofinjection can also be used such as intramuscular, subcutaneous or viainhalation. Oral administration can also be used as can transdermaldelivery or intraosseus delivery.

[0083] Respiratory Disorders

[0084] Bipolar trans carotenoid salts can be used to treat acute andchronic respiratory disorders. These are described as conditions inwhich the arterial partial pressure of oxygen is reduced, such as valueof 60 to 70 mm Hg rather than the normal value of 90-100 mm Hg. Suchacute and chronic respiratory disorders include emphysema, acute lunginjury (ALI), acute respiratory distress syndrome (ARDS), chronicobstructive pulmonary disease (COPD) and asthma.

[0085] TSC increases the value of the partial pressure of oxygen in theblood when it is low (this is a symptom of emphysema, ARDS and COPD).Increasing the partial pressure of oxygen in the blood relieves many ofthe symptoms of emphysema, ARDS and COPD. TSC does not cure the cause ofthe disease, but relieves the oxidative distress and damage resultingfrom that underlying cause.

[0086] Hemorrhagic Shock

[0087] Hemorrhagic shock is marked by a decrease in oxygen consumption.Bipolar trans carotenoid salts increase the body's oxygen consumption bycausing more oxygen to diffuse from the red blood cells to the tissues.TSC has been shown to increase the oxygen consumption of rats undergoinghemorrhagic shock, and has also been shown to offset other symptoms ofshock. The compounds of the invention cause the low blood pressure toincrease, reduce the increased heart rate, and reverse the bloodacidosis that develops during shock. The compounds of the invention alsoreduce organ damage subsequent to hemorrhagic shock.

[0088] The compounds of the invention can be used for hemorrhagic shockby administering them by inhalation, injecting them, or by adding themto a standard resuscitation fluid (Ringer's lactate or normal saline).

[0089] Cardiovascular Disease

[0090] In western culture, the leading cause of death is ischemic heartdisease. Death may result from either a gradual deterioration of theability of the heart to contract or, frequently, a sudden stoppage.Sudden cardiac death (SCD) covers the time period beginning 60 secondsafter symptoms begin to 24 hours later. These deaths are usually aconsequence of acute coronary occlusion (blockage) or of ventricularfibrillation (which can result from the occlusion).

[0091] Myocardial ischemia exists when there is an insufficient supplyof oxygen to the cardiac muscle. When coronary blood flow is extremelylow, cardiac muscle cannot function and dies. That area of muscle issaid to be infarcted. Most often, diminished coronary blood flow iscaused by atherosclerosis that occurs in the coronary arteries. Ischemiaresults in impaired mechanical and electrical performance and musclecell injury, which may lead to a lethal arrhythmia, called ventricularfibrillation (VF). In ventricular fibrillation, the electrical activityof the ventricles of the heart is chaotic and results in anelectrocardiogram with an erratic rhythm and no recognizable patterns.Ventricular fibrillation occurs frequently with myocardial ischemia andinfarction and is nearly always the cause of sudden cardiac death.Bipolar trans carotenoid salts are beneficial in treating myocardialischemia. Atherosclerosis, which is frequently a precursor to myocardialinfarction, and congestive heart failure can also be treated with thesesalts.

[0092] Ischemia

[0093] Bipolar trans carotenoid salts are also beneficial in treatingother forms of ischemia (insufficient blood flow to tissues or organs)such as kidney, liver, spinal cord, and brain ischemia including stroke.

[0094] Surgery

[0095] Surgery frequently involves either blood loss or clipping ofarteries (e.g., bypass surgery), which can cause ischemia. Bipolar transcarotenoid salts are beneficial as a pretreatment for surgery, or as atreatment during or after surgery.

[0096] Hypertension

[0097] Hypertension, or high blood pressure, is frequently associatedwith cardiovascular disease. The compounds of the invention can be usedto reduce blood pressure.

[0098] Performance Enhancement

[0099] BTCS enhance aerobic metabolism, increasing oxygen consumptionlevels during walking, running, lifting, etc. Endurance is alsoincreased.

[0100] Traumatic Brain Injury

[0101] Hypoxia following traumatic brain injury results in increasedbrain damage. BTCS increase oxygen levels in brain tissue after impactinjury (focal or diffuse injury). Examples of impact injury includecar/motorcycle accidents and falls. BTCS also augment the amount ofoxygen reaching normal brain tissue when hyper-oxygen therapy is used.

[0102] Alzheimer's Disease

[0103] BTCS increase brain oxygen consumption levels in Alzheimer'sDisease, thus alleviating symptoms of Alzheimer's Disease. Blood flowand oxygen consumption decline to level some 30% below that seen innon-demented elderly people Wurtman, Scientific American, Volume 252,1985.

[0104] The increased oxygen consumption levels in the brain created byBTCS also reduce memory loss.

[0105] Diabetes

[0106] BTCS are useful for treating complications of diabetes such asulcers, gangrene and diabetic retinopathy. Diabetic foot ulcers healbetter with hyperbaric oxygen breathing treatment, M. Kalani et al.Journal of Diabetes & Its Complications, Vol 16, No. 2, 153-158, 2002.

[0107] BTCS also help the complication of diabetic retinopathy which isrelated to low oxygen tension, Denninghoff et al., Diabetes Technology &Therapeutics, Vol. 2, No. 1, 111-113, 2000.

[0108] Other Uses

[0109] Bipolar trans carotenoid salts can also be used for the treatmentof spinal cord injury, cerebral edema, anemia, and skin papillomas. Inall cases, they alleviate the condition, making it less severe. It isbelieved that this results from the increase in oxygen consumption thatresults from the use of bipolar trans carotenoid salts.

[0110] Further, bipolar trans carotenoid salts can be used to increasediffusion of other physiologically important molecules such as glucose,CO₂ or NO. BTCS also scavenge oxygen-derived free radicals.

[0111] The following Examples are illustrative, but not limiting of thecompositions and methods of the present invention. Other suitablemodifications and adaptations of a variety of conditions and parametersnormally encountered which are obvious to those skilled in the art arewithin the spirit and scope of this invention.

EXAMPLES Example 1

[0112] Synthesis of Trans Sodium Crocetinate

[0113] Trans sodium crocetinate is synthesized by coupling a symmetricalC₁₀ dialdehyde containing conjugated carbon-carbon double bonds with[3-carbomethoxy-2-buten-1-ylidene]triphenylphosphorane. This product isthen saponified using a solution of NaOH/methanol.

[0114] To ethyl bromoacetate, trephenylphosphine dissolved in ethylacetate (at a concentration of around 2 moles/liter) is slowly added.After isolation, and treatment with base, the product can be treatedwith methyl iodide, followed by caustic, to form the phosphorane. Thebasic compound to form the carotenoid skeleton can be made starting witha ring compound such as furan in this case. Furan is reacted withbromine and methanol, followed by a selective deprotonation step to forma monoaldehyde. This is then coupled with the phosphorane. Acidicconditions deprotected the other dimethyl acetal group to afford thefree aldehyde. This compound is then reacted again with the samephosphorane to give the diethyl diester. The ester groups are reduced toalcohols, and subsequent oxidation (such as with MnO₂) results in theC₁₀ skeleton in the dialdehyde form. This is later reacted with aphosphorane made from tiglic acid. The tiglic acid is esterified withmethanol under acidic conditions to give the methyl ester, followed by abromination step. The resulting allylic bromide isomers are formed, andcan be separated using crystallization. Subsequent treatment of thedesired bromide with sodium hydroxide results in the desiredphosphorane. This phosphorane and the C₁₀ dialdehyde are then dissolvedin a solvent such as toluene or benzene and refluxed. The resultingproduct isolated as a powder and is then saponified with a 40%NaOH/methanol mixture to form the TSC after solvent removal.

[0115] Trans-sodium crocetinate 1 (TSC) was prepared in a 17 stepsynthetic sequence in an overall yield of 1.5%. A total of 4.1 g of TSCwas prepared with ethyl bromoacetate, furan and tiglic acid as startingmaterials.

[0116] Trans-sodium crocetinate (TSC) was synthesized fromsaponification of dimethyl crocetinate, the preparation of which wasbased on a total synthesis reported by Buchta and Andree.¹ The syntheticstrategy behind preparing dimethyl crocetinate was based on coupling thesymmetrical C₁₀ dialdehyde (2,7-dimethylocta-2,4,6-triene-1,8-dial) with(3-carbomethoxy-2-buten-1-ylidene)triphenylphosphorane.

[0117] Although the original Buchta and Andree article¹ was titled “TheTotal Synthesis of trans-2,2-Bisdimethyl-crocetin-dimethyl ester andtrans-Crocetin-dimethyl ester,” experimental details and yields were notreported. Procedures for the various steps leading to the C₁₀ dialdehydeand phosphorane were found after an extensive survey of the literature.Ultimately, TSC was prepared in a 17 step sequence with ethylbromoacetate, furan and tiglic acid as the starting materials in anoverall yield of 1.5%.

[0118] The C₁₀ symmetrical dialdehyde was prepared from ethylbromoacetate² and furan³ using Wittig chemistry. Ethyl bromoacetate wastreated with triphenylphosphine and methyl iodide to give thephosphorane 6:

a. TPP, EtOAc, 92%; b.1 N NaOH, CH₂; c. CH₃I, CH₂Cl₂; d.1 N NaOH,CH₂Cl₂.

[0119] The yield for the first step was a respectable 92%. Quantitationof the subsequent steps of this sequence were complicated by the natureof phosphorane 4 and phosphonium salt 5. Both of these compounds wereextremely viscous syrups which foamed vigorously while concentrating ona rotary evaporator. Both compounds could be conveniently handled asmethylene chloride solutions and the overall yield of phosphorane 6appeared to be acceptable from a qualitative point of view (estimated atbetter than 75%).

[0120] Furan was ring-opened with bromine to afford fumaraldehydebis(dimethylacetal) 8.³

e. Br₂, MeOH; Na₂CO₃, 77% f. Amberlyst 15, H₂O, acetone, 72%

[0121] Mono-deprotection of bis(dimethylacetal) 8 under acidicconditions⁴ gave aldehyde 9, which was then coupled with phosphorane 6to give 10 in a 45% yield. Acidic conditions were used to deprotect thedimethylacetal 10. Treating 11 with phosphorane 6 gave diester 12. Theester groups were reduced to alcohols by DIBAL-H and subsequentoxidation with MnO₂ gave the C₁₀ dialdehyde 14. The transstereochemistry of 14 was determined by NMR data. In particular, the C₂symmetry of the compound gave the expected 5 resonances in the ¹³C Nspectrum and the ¹H NMR spectrum showed signals at δ 9.54 (1H), 7.07(2H) and 1.95 (3H).

g. CH₂Cl₂, 45%; h. Amberlyst 15, H₂O, acetone, 42-65%; i. 6, CH₂Cl₂,50-81%; j. DIBAL-H hexanes, 75-81%; k. MnO₂, acetone, 26-58%.

[0122] The range in yields of steps h-k reflect improvements inisolation from intial pilot studies to scaled up reactions.

[0123] Tiglic acid 15 was converted to phosphorane 20 in a 4 stepsequence. Fisher esterification conditions on 15 gave the methyl ester16. Reaction with NBS gave a mixture of 59% methyl γ-bromotiglate, 26%methyl α-bromotiglate and the balance of the material was unreactedstarting material. The formation of regioisomers was expected based onthe reported literature.⁵ In the following step, the α/γ mixture ofphosphonium salts was recrystallized to give the desired γ-phosphoniumbromide 19.⁶ Subsequent treatment with sodium hydroxide gave thephosphorane 20.

I. H₂SO₄, MeOH, 42%; m. NBS, benzoyl peroxide, 59%; n. TPP, C₆H₆, 40%;o. NaOH, H₂O, 81%

[0124] Phosphorane 20 and C₁₀ dialdehyde 14 were coupled by refluxing inbenzene.⁶ Dimethyl crocetinate 21 was isolated as a red powder.Saponification of the methyl ester proved to be more difficult thanexpected. Treating the ester 21 with 2 eq. NaOH in THF/H₂O at r.t. andreflux left the material unchanged. Solubility appeared to be asignificant problem, so pyridine was added. While this did dissolve mostof the solids, refluxing a mixture of pyridine and 2.5 N NaOH yielded noproduct. Standard THF/2.5 N NaOH saponification conditions also had noeffect on the ester. Eventually, 40% NaOH/methanol at reflux for anovernight period proved to be successful. This gave TSC 1 as an orangesolid.

p. C₆H₆, reflux,.33-38%; q. MeOH, 40% aq. NaOH, 58-65%.

[0125] Attempts were made to dissolve TSC in order to obtain a ¹H NMRspectrum. However, TSC was practically insoluble in most common organicsolvents (chloroform, DMSO, pyridine, methanol, acetone, and glacialacetic acid). The TSC produced from this project was characterized byIR, UV, HPLC and elemental analyses. IR showed characteristic absorbanceat 1544 and 1402 cm⁻¹ (consistent with conjugated carboxylates). UV andHPLC were consistent with authentic TSC.⁷ Elemental analyses gavesatisfactory values.

[0126] The overall yield of the reaction sequence was 1.5% (based onfuran).

[0127] The synthesis is described in detail below:

[0128] All reagents and chemicals were purchased from Aldrich or Sigmaand used as received unless stated otherwise. Solvents were purchasedfrom Fisher Scientific as ACS reagent or HPLC grade and used withoutfurther purification. Anhydrous solvents were purchased from Aldrich inSure/Seal™ bottles and used directly without further purification.Deionized water was obtained from an in-house Culligan water treatmentsystem.

[0129] Melting points were obtained on a Mel-Temp II and wereuncorrected. Infrared spectra were measured on a Perkin-Elmer 1600 FFIRspectrophotometer. Nuclear magnetic spectra were measured on a JEOLFX90Q spectrometer using a 5 mm multinuclei probe with internal orexternal deuterium lock depending on the nature of the sample. Protonand carbon NMR chemical shifts were assigned relative to TMS or thedeuterated solvent respectively.

[0130] Phosphorus NMR spectra were generally run in the proton-decoupledmode with a coaxial insert tube of 5% aqueous phosphoric acid as theexternal standard.

[0131] Routine analyses by gas chromatography to evaluate reactionprogress or estimate product composition were conducted on a Varian 3700gas chromatograph equipped with a flame ionization detector and aHewlett Packard 3394A integrator. A 1 microliter solution was injectedonto a 15 meter DB5 column (0.53 mm ID and 1.5 micron film thickness)with helium carrier gas using a temperature program from 50 to 250° C.at 20° C./min with a 10 minute hold at 250° C. The injector and detectortemperatures were typically set at 250° C.

[0132] Thin layer chromatography was conducted on Baker-flex 2.5×7.5 cmsilica gel plates with or without fluorescent indicator (1B2 or 1B2-F)depending on the method of detection. The components on the developedplates were detected by UV.

[0133] Elemental analyses were conducted by Quantitative Technologies,Inc., Whitehouse, N.J.

[(Ethoxycarbonyl)methylene]triphenylphosphorane (4)² ACL-G29-1

[0134] Triphenyl phosphine (235.6 g, 0.90 mol) was dissolved in EtOAc(540 mL). Approximately 30 min was required for all of the solids todissolve. The process was endothermic (solution cooled to 13° C. whenthe ambient temperature was 20° C.). A solution of ethyl bromoacetate(100 mL, 0.90 mol) in EtOAc (400 mL) was added dropwise over a 1.5 hperiod. A white precipitate formed during the addition. Stirredovernight (20 h) at ambient temperature (18° C.).

[0135] The solids were collected by vacuum filtration rinsing withcopious amounts of Et₂O. Dried overnight in vacuo at 45° C. to give 3 asa white solid 356.3 g, 92.6% yield (0.83 mol). ¹H NMR was consistentwith literature values.

[0136] The solid was dissolved in methylene chloride (3 L) and treatedwith 1 M NaOH (3.6 L) in a 12 L flask with vigorous stirring for 45 min.The organic layer was separated and the aqueous phase was extracted withadditional methylene chloride (2×1 L). Organic layers were dried (MgSO₄)and concentrated until approximately 1 L of volume remained. A smallamount of material was removed and examined by ¹H NMR and found to beconsistent with literature values.

[1-(Ethoxycarbonyl)ethylidene]triphenylphosphoniun iodide (5)² ACL-G29-2

[0137] The material from ACL-G29-1 was treated with iodomethane (64.0mL, 1.03 mol) as the reaction flask was cooled in an ice bath. Thereaction mixture was checked by TLC (silica gel, 10% MeOH/CHCl₃) whenthe addition was completed (1 h) and it showed a considerable amount ofstarting material remained. The ice bath was removed and the reactionmixture was checked by TLC after 1.5 h, it looked complete based on atightening of the main band (s.m. streaked). The reaction mixture wasconcentrated on a rotary evaporator, when most of the solvent wasremoved, the product began foaming and creped up the vapor duct. Thephosphonium salt 5 appeared was an extremely viscous syrup which waskept as a methylene chloride solution to facilitate handling. Because ofthe nature of 5, the material was not quantitated.

[1-(Ethoxycarbonyl)ethylidene]triphenylphosphorane (6)² ACL-G29-2A

[0138] A portion of 5 dissolved in CH₂Cl₂ (350 mL) and vigorouslystirred with 1 M NaOH (500 mL) for 45 min. The organic layer wasseparated and the aqueous was extracted with CH₂Cl₂ (2×100 mL). Combinedorganic layers were dried (MgSO₄) and concentrated to give 6 as a yellowsolid, 8.0 g. ¹H NMR spectrum was consistent with literature values.

Fumaraldehyde bis(dimethylacetal) (8)³ ACL-G29-3

[0139] A solution of furan (88.0 g, 1.29 mol) in anhydrous MeOH (650 mL)was cooled to −45° C. under N₂. A solution of bromine (68.0 mL, 1.32mol) was added dropwise over a 2.5 h period at a rate to maintain ≦−45°C. The red solution was allowed to warm to −10° C. over a 2.5 h periodand held for an additional 2 h. The reaction mixture was a pale ambercolor. Addition of 5 g Na₂CO₃ produced a considerable amount ofoutgassing and a 4° C. exotherm. The reaction mixture was cooled withdry-ice and the remaining Na₂CO₃ (210 g total) was added over a 50 minperiod. After holding at −10° C. overnight (11 h, the cooling bath wasremoved and the reaction mixture was allowed to warm to room temperatureand stirred for 20 h.

[0140] The salts were removed by vacuum filtration and the filtrate wasvacuum distilled with a vigreux column until approximately 150 mL hadbeen removed. Additional salt had precipitated out and was causing thedistillation pot to bump violently. After filtration, another 150 mL wasdistilled and more salt came out of solution. Once again, severe bumpingwas a problem. The still pot was cooled, filtered, the filtrate treatedwith Et₂O (400 mL) and the precipitate removed by vacuum filtration. Atleast 120 g of salt was collected (early crops of salt were discardedwithout quantitation). The majority of the Et₂O was removed on a rotaryevaporator at 25° C. with a water aspirator. Distillation was resumedwith a vigreux column, 8 was collected as a clear, colorless liquid175.2 g (76.9% yield), b.p. 86-92° C./9 torr (lit. 85-90 C/15 torr). ¹HNMR spectrum was consistent for the desired product. GC analysis: 81.9%pure.

Fumaraldyhyde mono(dimethylacetal) (9)⁴ ACL-G29-4

[0141] Fumaraldyhyde bis(dimethylacetal) 8 (5.29 g, 0.03 mol) wasdissolved in acetone (120 mL). H₂O (1.80 mL) and Amberlyst 15 (1.20 g)were sequentially added. The mixture was stirred vigorously for 5 minthen filtered to removed the resin. During this time, the solutionturned from colorless to yellow. The filtrate was concentrated on arotary evaporator at room temperature and the light brown residue wasdistilled on a kugelrohr (37° C./200 millitorr) to give 9 as a yellowliquid, 2.80 g, 71.8% yield. A small amount of material was lost whenthe still pot bumped at the beginning. ¹H NMR spectrum was consistentfor the desired product, GC analysis indicated 80% purity.

ACL-G29-7

[0142] Fumaraldyhyde bis(dimethylacetal) 8 72.1 g, 0.41 mol) wasdissolved in acetone (1600 mL). H₂O (25.0 mL) and Amberlyst 15 (16.7 g,prewashed with acetone) was added. The mixture was stirred vigorouslyfor 5 min then filtered to removed the acid resin. The reaction mixturehad a slight yellow tint, much fainter than the previous large scaleprep. GC analysis indicated 34.5% product and 46.1% s.m. Treated withresin for another 5 min. GC analysis indicated 59.5% product and 21,7%s.m. Treated with resin for another 10 min (total time 20 min). GCanalysis indicated 73,9% product and 2.0% s.m. The filtrate wasconcentrated on a rotary evaporator at room temperature to give a brownoil, 54 g. Vacuum distillation gave a yellow-green oil, 34.48 g. GCanalysis indicated 64.7% purity (8.22 min) with a major impurity of17.5% (9.00 min) and 6.9% (9.14 min). Net recovered yield 22.3 g (0.17mol). Analysis of the forecut by GC showed extremely dirty material.

ACL-G29-13

[0143] Amberlyst 15 (8.61 g) was stirred in acetone (100 mL) for 30 minand collected by filtration. The acetal 8 (35.0 g, 0.16 mol) wasdissolved in acetonitrile (620 mL) and while mechanically stirred, acidresin and deionized H₂O (10.0 mL, 0.55 mol) was added. The course of thereaction was monitored by TLC (10:3 hexane:Et₂O), after 15 min most ofthe starting material had been converted. After 20 min, only a trace ofthe dimethyl acetal was detected. The resin was removed by filtrationand the filtrate was concentrated on a rotary evaporator at ≦40° C. Thecrude product was loaded on a Biotage column (7.5×9.0 cm) eluting with15% Et₂O in hexanes to give 19.8 g. 65% yield.

6,6-Dimethyoxy-2-methylhexa-2,4-dienoate (10)² ACL-G29-5

[0144] The ylide 6 (7.80 g, 22 mmol) was dissolved in methylene chloride(65 mL). A solution of fumaraldehyde mono(dimethylacetal) 9 (2.80 g, 17mmol) was added and the solution was stirred overnight. Solvent wasremoved at reduced pressure on a rotary evaporator. ¹H NMR of the crudeindicated desired product was present. Upon standing, crystals grew(presumably triphenylphosphine oxide). The solid (14.1 g after drying byvacuum filtration) was slurried in petroleum ether and filtered. Thefiltrate was concentrated to give a yellow oil with solids precipitatedout which was dissolve in methylene chloride (15 mL) and chromatographedon a Biotage 4×7.5 cm column eluting with methylene chloride to give 10as a yellow oil 1.8 g, 50% yield. ¹H NMR spectrum of the yellow oil wasconsistent literature values, however, a trace of methylene chlorideremained (0.75 eq) so the material was place on the rotary evaporatorfor 45 min. Mass was reduced to 1.5 g, 40.6% yield and the methylenechloride resonance disappeared. GC analysis major peak at 12.6 min,87.5% (50° C., 5 min hold, 20° C./min to 250° C. final temperature).

ACL-G29-6

[0145] A solution of ylide 6 (59.2 g, 0.16 mol) in methylene chloride(650 mL) was cooled in an ice bath and a solution of 9 (25.7 g, 0.19mol) was added. The solution was stirred overnight allowing the ice bathto melt. TLC (hexane:Et₂O 10:3) indicated at least 3 other compoundsrunning very close to the product. Examination of the aldehyde indicatedby GC analysis 50.0% purity. Solvent was removed to give a solid/oilmixture.

ACL-G29-8

[0146] Ylide 6 (59.2 g, 0.16 mol) and acetal 9 (0.19 mol) was coupled inmethylene chloride (1.1 L) and worked up as described above to give ayellow-green oil, 80 g. A portion of the crude reaction mixture (4.13 gof the original 80 g) was placed on the kugelrohr and distilled at 50°C./250 millitorr. A colorless oil was condensed 2.28 g, ¹H NMR indicatedit was the starting aldehyde while the product 10 remained in the stillpot, 1.85 g. Volatile components were removed from the bulk of the crudeproduct by kugelrohr distillation at 50° C./200 millitorr (net 35 g).

Ethyl 2-methyl-6-oxo-hexa-2,4-dienoate (11)² ACL-G29-9

[0147] Acetal 10 from the pilot still pot (ACL-G29-8, 1.85 g, 9 mmol)was dissolved in acetone (33 mL). Deionized H₂O (0.50 mL) and Amberlyst15 resin (0.35 g, prewashed with acetone) were added. The mixture wasstirred for 20 min. Filtered and concentrated on a rotary evaporator togive a yellow-green oil, 1.53 g. Chromatographed on a 4.5×7 cm Biotagecolumn eluting with 15% Et₂O in hexanes. This system gave incompleteseparation, but 0.32 g of the main component was isolated and analyzed;¹H NMR spectrum was consistent with literature data and IR (1711, 1682cm⁻¹) was consistent with the desired product. GC 95.6%. An additional0.35 g was recovered, although it was cross contaminated with less andmore polar material. The ¹H NMR spectrum indicated fairly cleanmaterial. GC 90.6%. Yield: 42%.

Diethyl 2,7-dimethylocta-2,4,6-triene-1,8-dioate (12)² ACL-G29-10

[0148] The aldehyde 11 (0.65 g, 3.5 mmol) from G29-9 was dissolved andmagnetically stirred in methylene chloride. Ylide (1.59 g, 4.4 mmol) wasadded. The light yellow-green solution turned a darker shade yellowwithin minutes. TLC after 10 min indicated starting material was almostcompletely consumed. After stirring for 20 h, the reaction mixture(brown solution) was filtered through a pipette partially filled withsilica gel. The filtrate was concentrated to give a brown solid. Thesolid was dissolved in 5% Et₂O in hexanes with a small amount of CHCl₃.Chromatographed on a 4×7.5 cm Biotage column eluting with 5% Et₂O inhexanes. The main product was isolated as a white crystalline solid, 045g, 50% yield. ¹H NMR spectrum was consistent with literature data.

ACL-G29-14

[0149] An additional amount of 12 was prepared as described above togive 21.8 g, 81.6% after chromatographic purification. ¹H NMR spectrumwas consistent with the desired product.2,7-Dimethylocta-2,4,6-triene-1,8-diol (13)² (ACL-G29-11)

[0150] The diester 12 (0.45 g, 1.8 mmol) was taken up in anhydroushexanes (15.0 mL). It appeared as though some of the material dissolved,but the mixture was quite cloudy. More material appeared to come out ofsolution when the mixture was cooled in a −78 C. bath. Neat DIBAL-H(2.50 mL) was dissolved in anhydrous hexanes (total volume 10.0 mL) anda portion (approximately 2 mL) was inadvertently siphoned into thereaction mixture as the diester was cooled in a dry-ice bath. Anadditional amount of DIBAL-H solution was added until a total of 5.0 mL(6.7 mmol) was added. The CO₂ bath was allowed to warm. After stirringfor 2 h 50 min, TLC indicated the diester was completely consumed. Bathtemperature was adjusted to −20° C. allowing to warm to 0° C. over 20min. Treated with H₂O/silica gel (2 mL/7 g) mixture for 30 min. AddedK₂CO₃ and MgSO₄. Filtered to remove the solids and thoroughly rinsedwith methylene chloride. Concentrated to give a white solid, 0.14 g, 50%yield. Note: TLC R_(f)=0.21 (5% MeOH/CHCl₃) is quite polar. Rinsing withmethylene chloride might not have been enough to recover all of theproduct. ¹H NMR spectrum was consistent with literature values.

ACL-G29-15

[0151] The diester (5.4 g, 21 mmol) was taken up in anhydrous hexanes(175 mL, poor solubility), cooled in a −78° C. bath and treated with asolution of DIBAL-H (14.5 mL in 50 mL anhydrous hexanes) over a 35 minperiod. Vigorous gas evolution was observed during the addition. Thecolor of the slurry went from white to dark yellow initially, thislightened up as additional DIBAL-H was added. Allowed to warm to −40° C.over 2 h, then transferred to a −28° C. bath overnight. The reactionmixture was treated with a homogeneous mixture of H₂O/silica gel (4mL/14.4 g) for 30 min. MgSO₄ (7.5 g) and K₂CO₃ (5.1 g) was added and thereaction mixture was removed from the cooling bath. Stirred 20 min, thenfiltered on a sintered glass funnel. The solids were washed withmethylene chloride—this caused a considerable amount of precipitate toform. Warming while placed on a rotary evaporator dissolved theprecipitated solids. The solids remaining on the sintered glass funnelwas washed with EtOAc (4×75 mL) and the filtrate was concentrated.

[0152] CH₂Cl₂ rinsings gave a pale-yellow solid, 1.7 g, ¹H NMR wasconsistent with literature values; EtOAc rinsings gave an off-whitesolid, 1.0 g, ¹H NMR consistent with literature values; total recover2.7 g, 75% yield.

ACL-G29-17

[0153] The diester (16.4 g, 6.5 mmol) was stirred in anhydrous hexanes(500 mL) under N₂ and cooled to −78° C. A solution of DIBAL-H (45 mL,253 mmol) in hexanes (150 mL) was added over a 1 h period. Allowed towarm to −30° C. and stirred overnight (17.5 h total time). A homogeneousmixture of H₂O/silica gel (12.3 g/43.7 g) was added and the mixture wasmanually swirled over a 45 min period. Added K₂CO₃ (15.5 g) and MgSO₄(23.5 g). Swirled over another 30 min period. Filtered on a sinteredglass funnel, rinsed with methylene chloride (ppt formed, presumablycaused by evaporative cooling) and the filtrate was concentrated. Thesolids were rinsed with several times with EtOAc (approximately 100 mLportions, 2 L total volume) and pooled with the original filtrate.Concentrated to give a yellow solid, 8.9 g, 81% crude yield. ¹H NMRspectrum was consistent with the desired product.

2,7-Dimethylocta-2,4,6-triene-1,8-dial (14)² ACL-G29-12

[0154] A slurry of MnO₂ (7.80 g, 90 mmol) was cooled in an ice bathunder N₂. A solution of diol 13 (0.14 g, 0.8 mmol) was added via pipetteas an acetone solution (5.0 mL). An additional 2.0 mL of acetone wasused to rinse the flask and complete the transfer. The ice bath wasallowed to melt overnight as the reaction mixture was stirred. Solidswere removed by filtration through Hyflo and concentrated to give ayellow solid. The material was dissolved in 10% Et₂O/hexanes with aminimal amount of CHCl₃ and applied to a column of silica gel (30×190mm) eluting with 10% E_(t)20/hexanes. The product could be followed as ayellow band as it eluted, 14 was isolated as a light yellow solid 37 mg,26% yield. ¹H NMR spectrum was consistent literature values.

ACL-G29-16

[0155] A solution of the diol 13 (2.70 g, 16 mmol) in acetone (500 mL)was cooled in an ice bath under N₂. MnO₂ (60.0 g, 0.69 mol) was added inportions over a 20 min period. The ice bath was allowed to melt as thereaction mixture was stirred overnight. The reaction mixture wasfiltered through Hyflo and the filtrate was concentrated to give ayellow solid, 1.6 g, 61% crude yield. ¹H NMR was consistent with theliterature values. The crude yellow solid was dissolved in methylenechloride (along with a small amount of 10% Et₂O in hexanes was added)and charged to a 4×7.5 cm Biotage silica gel column. Eluted initiallywith 10% ether in hexanes (1 L), then increased polarity to 15% Et₂O (1L) and 20% Et₂O (0.5 L). Recovered a yellow solid 1.0 g, 38% yield. ¹HNMR spectrum consistent with desired product.

ACL-G29-21

[0156] A solution of the diol (9.31 g, 60 mmol) in acetone (500 mL) wascooled in an ice bath under N₂. MnO₂ (100 g, 1.15 mol) was added and themixture was stirred as the ice bath was allowed to melt overnight.Checked by IR after 24 h, significant amount of product had formed, butstill quite a bit of alcohol present. Added an additional 50 g ofoxidant and continued stirring for another overnight period. A portionof the reaction mixture was filtered and checked by ¹H NMR, the reactionappeared complete based on the consumption of starting material. Therest of the reaction mixture was filtered through a pad of Hyflo andthoroughly rinsed with acetone. Concentrated to give a dark yellowsolid. Azeotroped once with 40 mL benzene then dried in vacuo at 40° C.for 5 h, then at r.t. overnight. Recovered 5.28 g, 58% yield. ¹H NMR andIR spectra were consistent for the desired product.

Methyl Tiglate (16)

[0157] In a 2L 3-neck flask fitted with an overhead stirrer, condenserand thermometer, a solution of tiglic acid 15 (89.8 g; 0.9 mol) and 5 mLconcentrated sulfuric acid (0.09 mol) in 900 mL methanol was heated atreflux for 20 hrs. The solution was cooled to 25° C. and the excessmethanol was stripped at 30° C. and 27 in Hg vacuum on a rotaryevaporator. GLC analysis of the recovered methanol distillate showedproduct in the overheads. The resulting two-phase, light brownconcentrate was taken up in 500 ml ethyl ether and washed successivelywith 250 mL water, 250 mL 10% aqueous sodium bicarbonate and 250 mLsaturated brine. The ether solution was dried over anhydrous potassiumcarbonate, filtered and stripped on the rotary evaporator at 25° C. and17 in Hg vacuum to give crude methyl tiglate as a near colorless oil;43.6 g (42% yield). GLC analysis showed one major volatile product witha retention time of 2.7 min compared to 3.8 min for the starting tiglicacid. Proton NMR in CDCl₃ showed the expected signals with some traceethyl ether contamination: 1.79 ppm (d, 3H), 1.83 (s, 3H), 3.73 (s, 3H),6.86 (q, 6.6 Hz). IR (neat on KBr): ester carbonyl at 1718 cm⁻¹. Thisoil was used as is in the next step.

Methyl γ-Bromotiglate (17)⁵

[0158] In a 1L 4-neck flask fitted with an overhead stirrer, athermometer and a condenser, a stirred mixture of the crude methyltiglate (43.6 g; 0.38 mol), N-bromosuccinimide (68 g; 0.38 mol) and 70%benzoyl peroxide (5.34 g; 0.015 mol) in 500 mL carbon tetrachloride washeated at reflux for two hours. After cooling to 20° C., the insolublesuccinimide (38.1 g 100% recovery) was suction filtered off. Thefiltrate was washed three times with 250 mL water, dried over MgSO₄ andthen stripped on a rotary evaporator at 25° C. and 26 in Hg vacuum togive a yellow oil; 78.8 g. Proton NMR of this oil in CDCl₃ gave acomplex spectrum. The methylene protons for the desired γ-bromo esterwere assigned to the doublet centered at 4.04 ppm (8.6 Hz), while thesame protons for the α-bromo isomer were ascribed to the singlet at 4.24ppm. Proton integration of these signals and the methyl multiplet from1.6 to 2.0 ppm suggested the following composition (mole %):

[0159] γ-bromo ester: 59%

[0160] α-bromo ester: 26%

[0161] starting material: 15%

[0162] This crude oil was used in the next step without any furtherpurification.

[0163] This reaction was also run on a 0.05 mole scale using only 0.87equivalents of N-bromosuccinimide under otherwise identical conditions.The composition of this crude oil was estimated based on its proton NMRspectrum as 52% γ-bromo ester, 24% α-bromo ester and 23% unreactedmethyl tiglate. GLC analysis of this oil was slightly more complicatedshowing other minor components.

Triphenylphosphonium Salt of Methyl γ-Bromotiglate (19)⁶

[0164] In a 2L 4-neck flask fitted with a thermometer, a 100 mL constantpressure addition funnel and a condenser connected to a static nitrogensystem, a stirred solution of the crude methyl γ-bromotiglate (78.8 g)in 350 ml benzene was treated dropwise with a soluton oftriphenylphosphine (95 g; 0.36 mol) in 350 mL benzene over a period of1.75 hrs. The temperature of the mixture exothermed slightly from 24 to27° C. under otherwise ambient conditions. After the addition, thereaction was stirred vigorously overnight to afford a slurry of whitesolid containing a yellowish gum that adhered to the walls of the flask.The white solid was suction filtered onto a sintered glass funnelwithout disturbing the yellowish gum. The flask was washed twice with100 mL benzene and poured onto the filter. The filter cake was washedwith 50 mL benzene and then twice with 50 mL hexane. The wet cake wasdried in a vacuum oven at ambient temperature for 5.5 hours. The driedwhite powder [93 g; mp=125° C. dec)] was dissolved in 150 mLacetonitrile with heat to give a clear yellow solution. Ethyl acetate(300 mL) was added to this hot solution and the product started tocrystallized after adding about 100 mL ethyl acetate. The flask wasstored in the refrigerator overnight. The product was suction filteredand washed with a minimum amount of 1:2 acetonitrile and ethyl acetate;45.0 g. mp=187-190° C. (dec). lit mp=183° C. (dec).

[0165] The gummy solids in the reaction flask were recrystallized from10 mL acetonitrile and 20 mL ethyl acetate. Also, additional solidsprecipitated overnight from the benzene mother liquor. These solids werefiltered and recrystallized in the same manner. Both samples wererefrigerated for 2 hours and suction filtered to give additonal product;13.3 g. The benzene filtrate was stripped on a rotary evaporator and theyellow oil taken up in 10 mL acetonitrile and precipitated with 20 mLethyl acetate. The slurry was stored in the refrigerator overnight togive additional product as a white solid; 4.6 g. m.p. 185-187° C. (dec).Total yield of the desired phosphonium salt as a white solid was 62.9 gor 36.2% yield based on the crude methyl tiglate. Proton NMR (CDCl₃,TMS) ppm 1.55 (d, 4Hz, 3H), 3.57 (s, 3H), 4.9 (dd, 15.8 & 7.9 Hz, 2H),6.55 (broad q, 6.6-7.9 Hz, 1H), 7.4-7.9 (m, 15H). Proton-decoupledPhosphorus NMR (CDCl₃, 5% aq H₃PO₄ coaxial external standard) 22.08 ppm.Partial Carbon NMR (CDCl₃): CO₂CH₃, (166.6 ppm, d, J_(CP)=3 Hz),olefinic CH (117.5 ppm, d, J_(CP)=86.1 Hz), CO₂CH₃, (52.0 ppm), Ph₃P—CH₂(25.4 ppm, d, J_(CP)=50.6 Hz) and CH₃ (13.4 ppm, d, J_(CP)=2.4 Hz).Partial IR (KBr pellet): ester carbonyl at 1711 cm⁻¹.

(3-Carbomethoxy-2-buten-1-ylidene)triphenylphosphorane (20)⁶

[0166] In a 5L 5-neck flask fitted with an overhead stirrer, an additionfunnel and a thermometer, a solution of sodium hydroxide (5.12 g; 0.128mol) in 250 ml water was added dropwise to a vigourously stirredsolution of the triphenylphosphonium salt of methyl y-bromotiglate (58.3g; 0.128 mol) in 2,500 mL water over a period of 41 minutes at 25° C.The yellow slurry was stirred for 10 minutes at room temperature andthen suction filtered. The filter cake was washed with 1,800 mL waterand then thoroughly dried on the filter with a nitrogen blanket. Theyellow solid was then dried overnight in a vacuum desiccator over P₂O₅at room temperature and 27″ Hg vacuum; 35.3 g (73.7% yield). mp=145-150°C. lit mp=145-165° C. Proton-decoupled phosphorus NMR in CDCl₃ showedtwo peaks at 17.1 ppm and 21.1 ppm in a ratio of 93:7. Proton NMR(CDCl₃, TMS) ppm 1.89 (s, 3H), 3.58 (s, 3H), 7.3-7.8 (m, 17H). A smallbut detectable singlet at 1.74 ppm was also apparent in this spectrumwhich was attributed to the impurity. This solid was used withoutfurther purification in the next step.

Dimethyl crocetinate (21)⁶ ACL-G29-18

[0167] The dialdehyde 14 (0.48 g, 2.9 mmol) was added to a 100 mL roundbottom flask. Benzene (20 mL) was added and the solids were dissolvedwith magnetic stirring. The ylide was added, an additional 10 mL benzenewas used to wash the compound into the flask. Warmed to a vigorousreflux for 6 h. The reaction mixture was allowed to cool overnight.Contrary to literature reports, a very small amount of solid had formed.The reaction mixture was concentrated, the residue was taken up in MeOH(30 mL) and boiled for 30 min. Upon cooling to ambient temperature, thesolids were collected by vacuum filtration. An NMR sample was preparedby dissolving 20 mg into 0.5 mL CDCl₃, somewhat surprisingly, thisrequired warming with a heatgun to dissolve completely. ¹H NMR spectrumwas recorded and found to be consistent with the desired product. Theremaining material was dissolved in hot benzene, filtered, the filtratewas concentrated, taken up in MeOH, cooled in an ice bath and solids redsolids were collected, 334 mg, 33% yield. This material did not appearto be any more soluble than the material which was originally isolated.

ACL-G29-18A

[0168] Dialdehyde 14 (5.78 g, 35 mmol) was dissolved in benzene (300 mL)under N₂. Ylide 20 (35.3 g, 94 mmol) was added and the mixture waswarmed to reflux for 6 h forming a dark red solution. After allowing thereaction mixture to cool overnight, red solids were collected by vacuumfiltration and rinsed with methanol. Transferred to a 500 mL RBF andrefluxed with approximately 65 mL methanol for 30 min. Cooled andcollected a red solid. Rinsed with cold methanol and dried in vacuo togive 21 as a red solid, 3.00 g. ¹H NMR and IR spectra were consistentwith the desired product.

[0169] The original filtrate (from the reaction mixture) wasconcentrated on a rotary evaporator and the dark residue was taken up in100 mL methanol and refluxed for 40 min. Cooled in an ice bath andcollected by vacuum filtration a red solid. Rinsed with cold methanoland dried in vacuo to give 21 as a red solid, 1.31 g. ¹H NMR spectrumwas consistent with the desired product.

[0170] The filtrates were pooled, concentrated and taken up in 75 mLmethanol and allowed to sit overnight at r.t. A red solid was recoveredby vacuum filtration: 0.38 g. ¹H NMR spectrum was consistent with thedesired product.

[0171] More solids had formed in the filtrate. Isolated by vacuumfiltration to give a red solid, 0.127 g. IR consistent with above. Totalrecovery: 4.89 g, 39% yield.

Saponification Attempt with THF/NaOH ACL-G29-19

[0172] A stirred suspension of diester 21 (100 mg, 0.28 mmol) in THF (2mL) and 1N NaOH (0.56 mL, 2 eq) was added. Stirred at r.t. overnight.TLC showed only starting material. Warmed to reflux, no change afterseveral hours. Added THF (6 mL) in an attempt to dissolve more of thesolids, but it didn't seem to matter. Continued refluxing overnight.Added more THF (about 6 mL, TLC showed only starting material), andrefluxed for another overnight period. Concentrated and check by ¹HNMR—only starting material (based on integration of the methyls andmethyl esters). Dissolved in pyridine (10 mL) while warmed on a heatingmantle. Added 2.5 N NaOH (1.0 mL). The dark orange solution turned deepred after several minutes. The heating mantle was removed, solids beganforming, mantle reapplied for 30 min, then stirred at r.t. overnight.Concentrated on high vacuum. The residue was insoluble in chloroform,DMSO, pyridine and sparingly soluble in H₂O. An IR (Nujol mull) showedC═O absorbance characteristic of the starting material.

Saponification with 2.5 N NaOH and THF ACL-G29-20

[0173] Diester 21 (37 mg, 0.10 mmol) was weighed into a flask andstirred in diethyl ether (4 mL). The solvent took on an orange color,but solids were still present. Added 1 mL of 2.5 N NaOH and warmed toreflux. After half an hour, most of the ether had evaporated. This wasreplaced with THF (3 mL) and refluxing was continued for several hours.Solid were collected by vacuum filtration, rinsed with deionized waterthen dried in a vacuum oven. IR showed only starting material.

Saponification with 40% NaOH (1) ACL-G29-22

[0174] Diester 21 (32 mg, 8.9 mmol) was weighed into a flask and stirredin methanol (1.5 mL). The solvent took on an orange/red color, butsolids were still present. Added 1.5 mL of 40% NaOH and warmed to refluxfor 17 h. After cooling to r.t., orange solids were collected by vacuumfiltration and rinsed with deionized water. Dried in vacuo at 40° C. togive 1 as an orange powder 21 mg, 59%. IR (KBr pellet) 3412, 1544, 1402cm⁻¹, the compound is probably hygroscopic, upfield carbonyl shift isconsistent with conjugation.

ACL-G29-22A

[0175] Repeated with 35 mg of diester 1 refluxing for 15 h. The reactionmixture was cooled in an ice bath, collected by vacuum filtration andwashed with cold deionized water. Dried in vacuo at 40° C. Recovered 1as an orange solid 25.5 mg, 65%.

ACL-G29-23

[0176] Diester 21 (0.48 g, 1.3 mmol) was taken up in methanol (15.0 mL)and 40% sodium hydroxide (15.0 mL) and warmed to reflux. Theheterogeneous red mixture turned orange after about 2 h. Heating wasdiscontinued after 6 h and the mixture was allowed to cool overnight. Anorange solid was collected by vacuum filtration and washed with colddeionized water. Drying in vacuo gave a friable orange solid, 0.36 g,68% yield.

ACL-G29-24

[0177] Diester 21 (1.10 g, 3.1 mmol) was placed in a 100 mL recoveryflask and heated to reflux in methanol (20 mL) and 40% NaOH (20 mL) for12 h. After cooling in an ice bath, an orange solid was collected byvacuum filtration and rinsed with deionized water. Drying in vacuo gave1.4 g, 100%. Anal Calcd for C₂₀H₂₂O₄Na₂-0.4H₂O: C, 63.29; H, 6.05; Na,12.11; H₂O 1.90. Found: C, 63.41; H, 6.26; Na, 11.75; H₂O, 1.93.

ACL-G29-25

[0178] Diester 21 (3.00 g, 8.4 mmol) was refluxed in methanol (80 mL)and 40% NaOH (60 mL) for 12 h. The product was isolated as an orangesolid as described above 2.7 g, 80%. Anal Calcd for C₂₀H₂₂O₄Na₂-0.4H₂O:C, 63.29; H, 6.05; Na, 12.1 1; H20, 1.90. Found: C, 63.20; H, 6.00; Na,11.93; H₂O, 1.81.Samples ACL-G29-23,-24 and -25 were ground on an agatemortar and combined as ACL-G29-A.

[0179] References

[0180] 1. E. Buchta and F. Andree Naturwiss. 1959, 46, 74.

[0181] 2. F. J. H. M. Jansen, M. Kwestro, D. Schmitt, J. LugtenburgRecl. Trav. Chim. Pays-Bas 1994, 113, 552.

[0182] 3. R. Gree, H. Tourbah, R. Carrie Tetrahedron Letters 1986, 27,4983.

[0183] 4. G. M. Coppola Syn. Commun. 1984, 1021.

[0184] 5. D. S. Letham and H. Young Phytochemistry 1971, 10, 2077.

[0185] 6. E. Buchta and F. Andree Chem. Ber. 1960, 93, 1349.

Example 2

[0186] Synthesis of Trans Potassium Norbixinate

[0187] Trans potassium norbixinate is synthesized by coupling asymmetrical C20 dialdehyde containing conjugated carbon-carbon doublebonds with [1-(ethoxycarbonyl)methylidene]triphenylphosphorane. Thepreparation of this compound is similar to that listed previously fortrans sodium crocetinate, except that the furan starting material isreplaced with the appropriate ringed structure. This product is thensaponified using a solution of KOH/methanol.

Example 3

[0188] Synthesis of a Longer BTCS

[0189] The above compound is synthesized by adding a symmetrical C₁₀dialdehyde containing conjugated carbon-carbon double bonds to an excessof [3-carbomethoxy-2-buten-1-ylidene]triphenylphosphorane. Thepreparation of this compound is similar to that listed previously fortrans sodium crocetinate, except that the furan starting material isreplaced with the appropriate ringed structure. The trans 40-carbonproduct is then isolated using a procedure such as chromatography. Thisproduct is then saponified using a solution of NaOH/methanol.

Example 4

[0190] TSC by Inhalation

[0191] TSC has been given to rats via an inhalation route. Ten rats weregiven TSC directly into the lungs. This was done by inserting a tubeinto the trachea, and nebulizing 0.2 ml of TSC solution (TSC dissolvedin dilute sodium carbonate solution) with about 3 to 6 mls of air. Forall dosages studied (0.5-2 mg/kg), about 20% of the drug was present inthe blood stream within one minute after it was given. For dosages of0.8 -1.6 mg/kg the drug was present in the blood stream for a period ofat least two hours.

Example 5

[0192] Improved Synthesis Method

Prep of Tetraethyl 2-Butenyl-1,4-bisphosphonate

[0193]

[0194] A 250 mL 3-neck flask was equipped with a Teflon-coatedthermocouple, a 60 mL constant pressure addition funnel and a simpledistillation head. Under a nitrogen atmosphere, neat triethyl phosphite(59 mL; 0.344 mol) was heated with a heating mantle controlled with aJKem controller at 140° C. A solution of trans-1,4-dichloro-2-butene(26.9 g; 0.215 mol) and triethyl phosphite (35 mL; 0.204 mol) was addeddropwise at 134-144° C. over a period of 93 minutes. The clear solutionwas then kept at 140° C. under nitrogen. After 37 minutes, gaschromatography of an aliquot (1 drop) in 1 mL of ethyl acetate showeddesired product, intermediate product and the two starting materials.

[0195] After 15.5 hrs at 140° C., gas chromatography of an aliquot (1drop in 0.5 mL EtOAc) showed the desired product with no detectablestarting dichloride or intermediate product. After 16 hrs, the faintyellow solution was cooled to room temperature under nitrogen. The faintyellow oil was distilled in a Kugelrohr with a two-bulb receiver and thefurther bulb cooled in a dry ice-acetone bath at 25-100° C. and 0.1-0.2torr to give a colorless oil (14.8 g) as a forecut. Gas chromatographyshowed only product in the Kugelrohr pot. This light amber oil wasdistilled in a Kugelrohr at 140° C. and 0.1-0.15 torr to give distillateas a colorless oil; 66.45 g (94.1 % yield). Gas chromatography showedonly one volatile component. GC-MS analysis showed that this componentwas the desired product, giving a small molecular ion at 328 m/z and abase ion at 191 m/z (loss of PO₃Et₂). Proton NMR was consistent with thedesired product. Carbon NMR also was consistent with the desiredbis(phosphonate diester), showing only long range (W-coupling) andnormal carbon-phosphorus coupling to the allylic carbon.

[0196] Pot residue—light yellow oil −0.8 g.

Prep of 1,1,8,8-Tetramethyoxy-2,7-dimethyl-2,4,6-ocatriene

[0197]

[0198] Under a nitrogen atmosphere, a magnetically stirred mixture oftetraethyl trans-2-butenyl-1,4-bisphosphonate (3.3 g; 10.0 mmol),pyruvic aldehyde dimethyl acetal (2.6 mL; 21.5 mmol) in 10 mL tolueneand 10 mL cyclohexane was treated successively with anhydrous potassiumcarbonate (10.2 g; 73.8 mmol) and powdered sodium hydroxide (1.25 g;31.2 mmol). The solution turned yellow immediately. The resulting slurrywas stirred at ambient temperature under nitrogen. The reaction slowlyexothermed, reaching a maximum of 38° C. after about 25 minutes. Also, agummy precipitated formed, which negatively impacted magnetic stirring.After 2.5 hrs, gas chromatography of an aliquot of the yellow-orangesolution (1 drop in 0.5 mL toluene) showed the two starting materialsand 3 other new components.

[0199] After 16.75 hrs at ambient temperature, gas chromatography of analiquot of the orange solution (1 drop in 0.5 mL toluene) showed only asmall amount of the starting bis(phosphonate diester). The resultingorange mixture with a gummy mass (unable to stir) was cooled in an icebath and quenched with 100 mL 10% aqueous NaCl. The solids weredissolved in this aqueous solution by working with a spatula. Themixture was then extracted with 200 mL 1:1 ether:hexane. The organiclayer was washed with 10% aqueous NaCl (200 mL) and then saturated brine(100 mL). The colorless organic layer was dried over Na₂SO₄. Gaschromatography showed three major components and no detectable startingbis(phosphonate diester). The thin layer chromatogram showed two majorspots and one minor spot. The Na₂SO₄ was suction filtered off and washedwith ether. The filtrate was concentrated on a rotary evaporator at 35°C. to give a colorless oil; 1.8 g. GC-MS Analysis showed that the threemajor volatile components were the isomeric products, giving molecularions at 256 m/z and base ions at 75 m/z [(MeO)₂CH⁺]. Proton NMR also wasconsistent with a mixture of isomeric products along with otherunidentified impurites. Yield of crude product =70.3%.

Prep of 1,1,8,8-Tetramethyoxy-2,7-dimethyl-2,4,6-ocatriene

[0200]

[0201] A mechanically stirred mixture of tetraethyltrans-2-butenyl-1,4-bisphosphonate (63.2 g; 0.19 mol), pyruvic aldehydedimethyl acetal (50 mL; 0.41 mol) in 200 mL toluene and 200 mLcyclohexane was treated successively with anhydrous potassium carbonate(196 g; 1.42 mol) and powdered sodium hydroxide (24.0 g; 0.60 mol). Thesolution turned yellow immediately. The resulting slurry was stirred atambient temperature under nitrogen. The reaction exothermed to 61° C.after about 11 minutes and the stirred mixture was cooled in an ice bathto drop the temperature to 35° C. After 4.7 hrs at 29-35° C., gaschromatography of an aliquot (3 drops in 0.5 mL toluene) showed nostarting bis(phosphonate). After ≈5 hrs, the mixture was cooled in anice bath to 13° C. and 10% aqueous sodium chloride (400 mL) was added asthe temperature rose to 30° C. More 10% aqueous sodium chloride (1,500mL) was added and the mixture was extracted with 3,000 mL 1:1ether:hexane. The tinted yellow organic layer was washed with 10%aqueous sodium chloride (2×1,000 mL) and then with saturated brine(1,000 mL). The tinted yellow organic layer was dried over Na₂SO₄,filtered and concentrated on a rotary evaporator at 30° C. to give alight yellow oil; 43.4 g. Gas chromatography showed three majorcomponents comprising 89% of the mixture with no detectable startingbis(phosphonate). TLC analysis showed one major and 3 minor components.

[0202] Proton NMR showed isomeric product plus toluene. The oil wasevaporated further on a Kugelrohr at 50° C. and 0.2 torr for 30 minutes;31.9 g. Proton NMR showed isomeric bis(acetal) product with nodetectable toluene.

[0203] Yield=65.5%

Prep of 2,7-Dimethyl-2,4,6-ocatrienedial at Higher Payload

[0204]

[0205] Under a nitrogen atmosphere, a magnetically stirred solution ofcrude 1,1,8,8-tetramethyoxy-2,7-dimethyl-2,4,6-ocatriene isomers (31.9g; 124.4 mmol) in tetrahydrofuran (160 mL), water (80 mL) and glacialacetic acid (320 mL) was heated at 45° C. with a heating mantlecontrolled with a JKem controller via a Teflon-coated thermocouple (9:03am). After 30 minutes, the mixture exothermed to a maximum of 54° C. andthen returned to the 45° C. setpoint. Gas chromatography of an aliquot(3 drops in 0.5 mL THF) after 3 hours showed some residual startingmaterial, two major and one minor product. The yellow reaction solutionwas cooled in an ice bath to 21° C. and then diluted with 4:1ether:dichloromethane (2,000 mL). This solution was then washedsuccessively with 20% aqueous NaCl (2,000 mL×2), 4:1 20% aq NaCl: 1Maqueous NaOH (2,000 mL×3)¹ and 20% aq NaCl (1,000 mL×2). The yelloworganic layer was dried over MgSO₄, filtered and concentrated on arotary evaporator to give a yellow solid; 18.9 g. Gas chromatographyshowed one major and one minor component starting bis(acetal). TLCanalysis showed one major spot and several minor, more polar impurities.This solid was dissolved in 250 mL refluxing methanol, cooled to roomtemperature and then in an ice bath for 1 hr. The slurry was suctionfiltered to give a yellow fluffy needles; 14.15 g. Gas chromatographyshowed 95:5 mixture of isomeric dialdehydes. This solid wasrecrystallized again with 200 mL refluxing methanol, cooled to roomtemperature and then in the refrigerator overnight.

[0206] The slurry was suction filtered and washed with freezer-chilledmethanol to give yellow needles; 11.2 g. Gas chromatography showed 97:3mixture of isomeric dialdehydes. TLC analysis showed one spot. Theneedles were dried in a vacuum oven at 45° C. for 160 minutes untilconstant weight; 10.75 g.

[0207] uncorrected mp=154-156° C. lit² mp=161-162° C. Proton NMR andCarbon NMR were consistent with the desired symmetrical dialdehyde.

[0208] The two methanol filtrates from the recrystallizations werecombined. The thin layer chromatogram showed product plus otherimpurities. The filtrates were concentrated and various crops collectedas shown below. Crop Appearance Amt (g) Isomeric Ratio 2 yellow powder1.4 80:20 3 yellow needles 2.6 75:25 4 yellow solid  4.45 46:30

[0209]

[0210] Crop 2 & 3: These combined crops were dissolved in 20 mLrefluxing ethyl acetate, cooled to room temperature and then in thefreezer for 1 hr. The slurry was suction filtered and washed withfreezer-chilled ethyl acetate to give yellow needles; 1.95 g. Gaschromatography showed 86:14 mixture of isomers. This solid wasrecrystallized again in ethyl acetate (10 mL) to give yellow needles;1.55 g. Gas chromatography showed 92:8 ratio of isomers. A thirdrecrystallization from ethyl acetate (10 mL) afforded yellow needles;1.25 g. mp=152-154° C. Gas chromatography showed 96:4 isomer ratio.Proton NMR confirmed as the desired dialdehyde. GC-MS analysis wasconsistent with the desired dialdehdye, showing a prominent M⁺ ion at164 m/z and a base ion at 91 m/z.

[0211] The ethyl acetate filtrate was combined with the yellow solidfrom the methanol filtrate (crop 4) and concentrated on a rotaryevaporator to give a yellow solid; 6.0 g. Gas chromatography showed a53:34 mixture of the two isomers along with other impurities.

[0212] The solid was dissolved in 100 mL dichloromethane and Davisilgrade 643 silica gel (33.5 g) was added. The mixture was stripped on arotary evaporator at 35° C. The silica gel with adsorbed material wasthen added to the sample introduction module for the Biotage system,which already contained a plug of glass wool and a layer of sand. Thesilica gel was then topped with filter paper. The Biotage 75S column waspreviously wetted with the solvent mixture with a radial compression of35 psi and solvent pressure of 20 psi. The column was eluted with 85:15hexane:ethyl acetate (6,000 mL). A void volume of 1,000 mL including theprewet stage was taken. Fractions of 250 mL were collected and combinedbased on thin layer chromatogram analysis. These fractions wereconcentrated on a rotary evaporator at 35° C. as shown below. FractionContent Appearance Amt (g) Comment 1 blank 2-3 A 4 tr A  5-10 B yellowsolid 3.9 Product Cut 11-18 tr B or tr C No evidence of close elutingimpurity 19-20 tr B or C & D

[0213] Fractions 5-10: The yellow solid was slurried in hexane andsuction filtered to give a bright yellow solid; 2.5 g. Gaschromatography showed an mixture of dialdehyde isomers in a ratio of67:33.

[0214] Total yield of 96-97% E,E,E-dialdehyde=10.75+1.25=12.0 g (58.8%yield).

Isomerization of 2,7-Dimethyl-2,4,6-ocatrienedial withpara-Toluenesulfinic Acid

[0215]

[0216] Under a nitrogen atmosphere, the 2:1 isomeric mixture of2,7-dimethyl-2,4,6-ocatrienedial and its off-isomer (2.5 g; 15.2 mmol)and 4-toluenesulfinic acid (0.35 g; 2.2 mmol) and 50 mL anhydrous1,4-dioxane was heated at reflux for 15 minutes. An aliquot (7 drops)was diluted in 0.5 mL 4:1 ether:dichloromethane and dried over K₂CO₃.Gas chromatography showed a 91:9 mixture of desired isomer andoff-isomer.

[0217] After cooling overnight at room temperature, the resulting slurrywas dissolved in 100 mL 4:1 ether:dichloromethane and washedsuccessively with water (50 mL×3), 0.2M aqueous NaOH (50 mL), water (50mL×2) and saturated brine (50 mL×3). After separation of the layers, theremaining rag layer was dissolved in dichloromethane. The combinedorganic layers were dried over MgSO₄, filtered and concentrated on arotary evaporator at 40° C. to give an orange solid; 2.2 g. Gaschromatography showed 93:7 ratio of desired dialdehyde to off-isomer.This solid was slurried in hexane and suction filtered to give an orangesolid; 2.15 g. This solid was recrystallized from 20 mL refluxing ethylacetate by cooling to 30-40° C. and then in the freezer for 1 hr. Theslurry was suction filtered and washed with freezer-chilled ethylacetate to give yellow-orange needles; 1.65 g. mp=158-160° C. litmp=161-162° C. Gas chromatography showed 96:4 ratio of desireddialdehyde to off-isomer. Proton NMR and Carbon NMR were consistent withthe desired dialdehdye isomer.

[0218] Yield=66%

[0219] Scaleup Prep of Methyl Tiglate with Thionyl Chloride in Methanol

[0220] A mechanically stirred solution of tiglic acid (397.35 g; 3.97mol) in 3,000 mL methanol was treated dropwise with neat thionylchloride (397 mL; 5.44 mol) over a period of 130 minutes as thetemperature climbed from 14° C. to a maximum of 50° C. after 80 minuteswith no external cooling. Gas chromatography of an aliquot showedcomplete conversion to the ester with no detectable tiglic acid. Afterstirring at ambient temperature for 1 hr, the solution was distilled atatmospheric pressure through a silvered, vacuum jacketed Vigreux column(400 mm×20 mm). The condensate was collected at mainly 57-61° C. with apot temperature of 58-63° C.; 630 mL in 2 hrs. Gas chromatography showedsignificant methyl ester in the distillate.

[0221] The Vigreux column was swapped with a less efficient column (30×2cm w/less indentations) to speed up the rate of distillation. At a pottemperature of 69-71° C., distillate was collected with a headtemperture of 65-69° C.; 1,300 mL over 2.25 hrs. Gas chromatographyshowed significant methyl ester in the distillate. The atmosphericdistillation was continued until the pot temperature reached 87° C.,distillate was collected during this period at a head temperture of69-83° C.; 975 mL over 2 hrs. Gas chromatography showed significantlymore methyl ester in the distillate than earlier fractions.

[0222] The yellow two-phase mixture in the pot was extracted with ether(300 & 200 nm), dried over K₂CO₃, filtered and concentrated on a rotaryevaporator at 25° C. to give an orange oil; 132.6 g (29.3% yield). Gaschromatography showed product. Proton NMR and carbon NMR were consistentwith the desired product with trace ethyl ether. Gas chromatography ofthe ether condensate showed some methyl ester in the overheads.

[0223] Distillate 3: The third methanol distillate (975 mL) wasconcentrated on the rotary evaporator at 25° C. to give a two phasemixture (100-150 mL). This mixture was extracted with ether (100 & 50mL), dried over K₂CO₃.

[0224] Distillate 2: The second methanol distillate (1,300 mL) wasconcentrated on the rotary evaporator at 25° C. to give a two phasemixture (30-50 mL). This mixture was extracted with ether (2×50 mL),dried over K₂CO₃.

[0225] The concentrated ether extracts for distillate 2 and distillate 3were combined, suction filtered and concentrated on a rotary evaporatorat 25° C. to give a colorless oil; 77.3 g.

[0226] Proton NMR and carbon NMR matched previous spectra of the desiredmethyl ester.

[0227] Total Yield=132.6+77.3=209.9 g (46.3%).

[0228] Alternatively, 1) methyl tiglate is commercially available fromAlfa, Lancaster or Acros. and 2), pilots can be run to make phosphoniumsalt via JOC, 64, 8051-8053 (1999).

[0229] Bromination of Methyl Tiglate

[0230] A mechanically stirred slurry of methyl tiglate (209.9 g; 1.84mol) and N-bromosuccinimide (327.5 g; 1.84 mol), 70% benzoyl peroxide(3.2 g; 0.009 mol) in 2,000 mL carbon tetrachloride was heated to reflux(78-81° C.) with a 1 L Kugelrohr bulb between the 5 L reaction flask andthe reflux condenser. After 2 hrs, reflux was stopped, the mantledropped and the stirrer shutoff. All of the solids floated on the CCl₄solution, suggesting succinimide with negligible NBS. The slurry wascooled in an ice bath to 20° C. and suction filtered to give an offwhitesolid; 180.7 g. No wash. The yellow filtrate was washed with water (1L×3), dried over MgSO₄. Gas chromatography showed starting methyltiglate and the two monobromides in ≈1:2:1 ratio along with other minorcomponents.

[0231] After filtering off the MgSO₄, the light yellow filtrate wasconcentrated on a rotary evaporator at 35° C. to give a light yellowoil; 327.1 g. Proton NMR and gas chromatography suggested the followingcomposition: Component NMR (mole %) GC (Area %) γ-Bromo 50% 49% α-Bromo26% 21% α,γ-Dibromo (?)  7%  4% Methyl Tiglate  6% 10% Other 11% —

[0232] Yield of desired product adjusted for 50% assay =46.0%

[0233] This oil is used as is in the next step.

[0234] Scaleup Reaction of Methyl γ-Bromotiglate with Triphenylphosphinein Acetonitrile with Slightly Higher Payload

[0235] Under a nitrogen atmosphere in a 5 L, 4-neck flask, the crudemixture of methyl γ-bromotiglate (322.6 g; 85% allylic bromide; 1.42mol) in 1,300 mL anhydrous acetonitrile was stirred mechanically.

[0236] A solution of triphenylphosphine (387.0 g; 1.48 mol) in 2,000 mLethyl acetate was added dropwise over a period of 4 hours. During theaddition, the temperature climbed from 22° C. to a maximum of 30° C.after adding about 40% in the first 75 minutes. After adding 60% of thetriphenylphosphine solution over 120 minutes, the solution became cloudyand continued to precipitate solids through the rest of the addition.After the addition, the funnel was rinsed with ethyl acetate (600 mL)and chased into the reaction mixture. The cream slurry was stirrred atambient temperature over the weekend.

[0237] The white slurry was suction filtered and the cake was washedwith 2:1 ethyl acetate:acetonitrile (150 mL×3). The white solid (352.55g) was dried in a vacuum oven at 40° C. for 4 hrs (constant weight after2 hrs); 322.55 g. mp=187-188° C. (dec). lit mp=183° C. (dec). Proton NMRand Carbon NMR matched previous spectra for the desired phosphoniumsalt. LC-MS analysis showed one major component, whose electrospray massspectrum in the positive mode was consistent with the desiredphosphonium salt giving a molecular ion at 375 m/z. Phosphorus NMRshowed a single phosphorus signal at 22.0 ppm.

[0238] Yield based on starting methyltiglate=100×322.55/(455.32×1.84×322.6/327.1)=39.0%

Prep of (3-Carbomethoxy-2-Z-buten-1-ylidene)triphenylphosphorane

[0239]

[0240] A mechnically stirred slight slurry of(3-carbomethoxy-2-E-buten-1-ylidene)triphenylphosphonium bromide (154.8g; 0.34 mol) in 3,400 mL deionized water was treated dropwise with asolution of sodium hydroxide (13.6 g; 0.34 mol) in 500 mL water at 23°C. over a period of 32 minutes with no obvious exotherm, but immediateprecipitation of a bright yellow solid. After stirring for 15 minutes,the bright yellow slurry was suction filtered, washed with water (1,500mL) and sucked dry to give a canary yellow solid; 151.7 g. This solidwas dried in a vacuum oven at 35-45° C. (3:50 pm) overnight.

[0241] After drying in the vacuum oven at 35-45° C. for 22.5 hrs, aconstant weight was obtained; 107.8 g. mp=144-160° C. lit mp=145-165° C.Proton NMR was similar to the previous spectrum of the desired ylideconsidering the differences in NMR field strength. Carbon NMR showed themethyl carbon's at 50.2 and 11.8 ppm with a complex aromatic region andno obvious signals for the olefinic carbons and the ylide carbon.

[0242] Yield=84.7%

[0243] Pilot Prep of Dimethyl Crocetinate

[0244] Under a nitrogen atmosphere, a magnetically stirred mixture of(3-carbomethoxy-2-Z-buten-1-ylidene)triphenylphosphorane (12.8 g; 34.2mmol) and 2,7-dimethyl-2,4,6-ocatrienedial (2.1 g; 12.8 mmol) in benzene(128 mL) was heated to reflux for 6 hrs using a timer.

[0245] The resulting slurry was cooled in an ice bath for 40 minutes,suction filtered, washed with benzene and sucked dry to melt the frozenbenzene to give a red solid; 2.1 g. TLC analysis showed a single, yellowspot. This solid was dried in a vacuum oven at 40-45° C. for 70 minutes;1.85 g (40.5% yield). uncorrected mp=210-213° C. lit³ mp=214-216° C.Proton NMR was similar to the pre spectrum of dimethyl crocetin on 90MHz instrument. Carbon NMR showed all 11 unique carbon signals with thecorrect chemical shift for the desired dimethyl ester with one minorimpurity signal that may be residual benzene. Electrospray mass spectrumsuggested decomposition and recombination of fragments.

[0246] TLC analysis showed that the red filtrate contained additionalproduct, triphenylphosphine oxide and an orange component with an R_(f)slightly lower than the isolated solid. The red filtrate wasconcentrated on a rotary evaporator at 35° C. to give red solids; 13.2g. This solid was heated at reflux in methanol (25 mL). The resultingslurry was then cooled in an ice bath, suction filtered after 60 minutesand washed with methanol to give a red solid; 0.6 g. This solid wasdried in the vacuum oven at 45° C. 135 minutes; 0.5 g. mp=203-208° C.Proton NMR showed desired diester with residual impurities. Carbon NMRshowed only signals for desired product. TLC analysis showed streakyproduct spot.

[0247] Filtrate was concentrated and saved.

[0248] Second Prep of Dimethyl Ester of Crocetin

[0249] Under a nitrogen atmosphere, 2,7-dimethyl-2,4,6-ocatrienedial(11.95 g; 12.8 mmol) was added in one portion to a mechanically stirredslurry of (3-carbomethoxy-2-Z-buten-1-ylidene)triphenylphosphorane (73.0g; 195.0 mmol) in 400 mL benzene and then chased with 330 mL benzene.The resulting brown slurry was heated to reflux for 6 hrs using a timerand cooled to room temperature overnight under nitrogen.

[0250] The resulting slurry was cooled in an ice bath to 6-10° C.,suction filtered and washed with benzene (50 mL×2) to give a red solid;10.05 g. TLC analysis showed a single yellow spot. This solid was driedin a vacuum oven at 40° C. (9:00 am) for 3.5 hrs with no weight loss;10.05 g (38.7% yield). mp=211-214° C. lit mp=mp=214-216° C. Proton NR anCarbon NMR matched the previous spectra for the desired dimethyl esterof crocetin.

[0251] The red filtrate was concentrated on a rotary evaporator at 40°C. to give a red solid; 84.4 g. TLC analysis was similar to the pilotrun. This solid was slurried in 165 mL methanol at reflux with magneticstirring. The resulting slurry was then cooled in an ice bath for 2.5hrs, suction filtered and washed with a minimal amount of methanol togive an orange paste; 10.5 g. TLC analysis showed a single, yellow spot.This paste was dried in a vacuum oven at 45° C. for 190 minutes; 5.6 g.mp=201-208° C. NMR showed desired diester with unknown aromaticimpurities.

[0252] This impure solid and two other similar solids from earlier runstotaling 6.5 g were dissolved in refluxing chloroform (75 mL) anddiluted with methanol and cooled in the refrigerator overnight.

[0253] The slurry was suction filtered and washed with a minimal amountof methanol to give red crystalline solid; 6.1 g. This solid was driedin the vacuum oven at 45° C. for 3 hrs until constant weight; 4.25 g.mp=211-213° C. Proton NMR and carbon NMR showed other olefinic oraromatic impurities. The solid was dissolved in refluxing toluene (150mL) and eventually cooled in the refrigerator for 130 minutes. Theslurry was suction filtered and washed with tolene to give a red solid;2.05 g. This solid was dried in the vacuum oven at 45° C. for 50 minuteswith no weight change; 2.05 g. mp=214-216° C. Proton NMR showed thedesired dimethyl crocetin with some residual toluene and negligibleoff-isomer impurities. Carbon NMR showed the desired dimethyl crocetinwith no detectable off-isomer impurities and 2-3 new residual signalsthat were consistent with toluene. Yield=45.5%.

[0254] Prep of Disodium Salt of Crocetin

[0255] A mechanically stirred slurry of dimethyl crocetin (13.95 g; 39.1mmol) and 40 wt % aqueous sodium hydroxide (273 mL; 3.915 mol) andmethanol (391 mL) was heated at reflux at 74° C. for 12 hrs using atimer.

[0256] The orange slurry was suction filtered through a Buchner funnelwith filter paper and a sintered glass funnel. Slow filtration.⁴ Theslurry in the sintered glass funnel was added to the solids in theBuchner funnel. The orange paste was washed with water (100 mL×3) andthen with methanol (50 mL×3). The orange paste was dried in a vacuumoven at 45-50° C.

[0257] After 21 hrs, the orange clumps weighted 24.25 g. The materialwas pulverized with a spatula and dried in the vacuum oven at 45-50° C.

[0258] After a total of 65.5 hrs of drying, amount of orange powder was23.1 g. The infrared spectrum showed extra bands compared to thereported IR spectrum of TSC, especially large bands at 3424 and 1444cm⁻¹. Proton NMR showed no evidence of methyl esters. However, theintegration of the olefinic and methyl regions were off, possibly due tophasing problems.

[0259] Assuming that the excess weight was due to sodium hydroxide, theorange solid was stirred magnetically in 400 mL deionized water for 35minutes. The slurry was suction filtered and the cake washed withdeionized water (50 mL×2) to give an orange paste. This material wasdried in a vacuum oven at 45-50° C. until constant weight. After about 7hrs, the solid was crushed and pulverized and dried further in thevacuum oven at 45° C. overnight.

[0260] After 21 hrs of drying at 45° C., amount of solid was 13.25 g.After further pulverizing and drying in the vacuum oven at 45° C.,amount of solid was 13.15 g. The infrared spectrum was consistent withthe reported IR spectrum. Proton NMR gave a proton NMR spectrum that wasconsistent with The disodium salt. HPLC analysis showed one majorcomponent with possibly one minor impurity. The electrospray negativeion mass spectrum of the major component was consistent with the desireddisodium salt of crocetin. Carbon NMR showed all ten unique carbonsignals for disodium salt of crocetin, verifying the symmetry of themolecule.

[0261] The original filtrate of water, sodium hydroxide and methanolprecipitated more solids during the water wash. This slurry was suctionfiltered, washed with water to give an orange paste. This paste wasdried in the vacuum oven at 45° C. for 18.5 hrs to give an orange solid;0.65 g. The spectral data were consistent with the desired disodium saltof crocetin. This solid was combined with the first crop.

[0262] Yield=13.15+0.65=13.8 g (94.8%).

[0263] Elemental Analyses of the first crop showed unacceptable valuesfor the desired product, suggesting sodium hydroxide contamination ofthe disodium salt of crocetin.

[0264] Water Wash of Disodium Salt of Crocetin

[0265] The disodium salt of crocetin (13.6 g) was slurried in 150 mLdeionized water and stirred magnetically at room temperature for 1 hr.The slurry was suction filtered onto a Buchner funnel. The orange pastewas then washed with water and the pH of the orange filtrate monitored.

[0266] The orange paste was sucked dry on the filter with a rubber dam.This paste was dried in a vacuum at 25-55° C. for 5.5 hrs to give afriable orange solid; 11.2 g. This solid was pulverized, transferred toa bottle and dried in the vacuum oven at 45° C. overnight.

[0267] Amount=11.1 g. Recovery=81.6%. The IR and Proton NMR spectramatched previous IR and proton NMR spectra of the desired disodium saltof crocetin. HPLC analysis showed a single component at 420 nm, whoseelectrospray mass spectrum in the negative ion mode was consistent withcrocetin.

[0268] Carbon NMR showed all ten unique carbon signals with the correctchemical shifts for the desired disodium salt of crocetin. Elementalanalysis gave acceptable data for the desired product.

[0269] References

[0270] 1. Tetrahedron Letters, 27, 4983-4986 (1986).

[0271] 2. F. J. H. M. Jansen, M. Kwestro, D. Schmitt & J. Lugtenburg,Recl. Trav. Chem. Pays-Bas, 113, 552-562 (1994) and references citedtherein.

[0272] 3. J. H. Babler, U.S. Pat. No. 4,107,030, Apr. 21, 1992.

[0273] 4. T. W. Gibson & P. Strassburger, J. Org. Chem., 41, 791 (1976)& J. M. Snyder & C. R. Scholfield, J. Am. Oil Chem. Soc., 59, 469(1982).

Example 6

[0274] Purity Determination of TSC Made According to the ImprovedSynthesis Method

[0275] For the TSC material synthesized according to the method ofExample 5, the ratio of the absorbance at 421 mn to the absorbance at254 nm was 11.1 using a UV-visible spectrophotometer.

Example 7

[0276] Oral Administration of TSC

[0277] TSC has been shown, in rats, to be absorbed into the blood streamwhen administered orally (via a gavage technique). In two rats, it wasfound that 1 to 2% of the dosage given was present in the blood streamat a time of 15 to 30 minutes after being given. The maximum amountabsorbed orally actually occurred earlier than that time.

[0278] It will be readily apparent to those skilled in the art thatnumerous modifications and additions can be made to both the presentcompounds and compositions, and the related methods without departingfrom the invention disclosed.

What is claimed is:
 1. A compound having the structure: YZ-TCRO-ZYwhere: Y=a cation Z=polar group which is associated with the cation, andTCRO=trans carotenoid skeleton wherein said compound is not TSC.
 2. Acompound as in claim 1 wherein Y is a monovalent metal ion selected fromthe group consisting of Na⁺, K⁺, Li⁺, or an organic cation selected fromthe group consisting of R₄N⁺, R₃S⁺, where R is H, or C_(n)H_(2n+1) wheren is 1-10.
 3. A compound as in claim 1 wherein Z is selected from thegroup consisting of a carboxyl (COO⁻) group, a sulfate group (OSO₃ ⁻) ora monophosphate group (OPO₃ ⁻), (OP(OH)O₂ ⁻), a diphosphate group,triphosphate or combinations thereof.
 4. A compound as in claim 1wherein TCRO is conjugated carbon-carbon double and single bondscontaining carbon atoms wherein the 4 single bonds which surround acarbon-carbon double bond all lie in the same plane and said compound islinear.
 5. A compound as in claim 1 wherein TCRO is

where X which can be the same or different, is H, a linear or branchedgroup having 10 or less carbons optionally containing a halogen, or ahalogen.
 6. A compound as in claim 1 wherein TCRO is

where X which can be the same or different, is H, a linear or branchedgroup having 10 or less carbons optionally containing a halogen, or ahalogen
 7. A compound as in claim 1 wherein TCRO is

where X which can be the same or different, is H, a linear or branchedgroup having 10 or less carbons optionally containing a halogen, or ahalogen.
 8. A compound as in claim 1 wherein TCRO is

where X which can be the same or different, is H, a linear or branchedgroup having 10 or less carbons optionally containing a halogen, or ahalogen.
 9. A method of solubilizing a BTCS having the structureYZ-TCRO-ZY where: Y=a cation Z=a polar group which is associated withthe cation, and TCRO=trans carotenoid skeleton comprising the steps of:a) preparing a dilute solution of sodium carbonate or sodiumbicarbonate, b) adding said dilute solution to deionized water to raisethe pH to 7 or above, c) adding a BTCS to the solution of step b).
 10. Amethod of solubilizing a BTCS having the structure YZ-TCRO-ZY where: Y=acation Z=a polar group which is associated with the cation, andTCRO=trans carotenoid skeleton comprising the steps of: a) adding a BTCSto a saline solution, b) removing undissolved material.
 11. A method ofsolubilizing a BTCS having the structure YZ-TCRO-ZY where: Y=a cationZ=a polar group which is associated with the cation, and, TCRO=transcarotenoid skeleton. comprising the steps of: a) adding a base to waterto make a basic solution, b) adding a BTCS to said solution.
 12. Amethod of solubilizing a BTCS having the structure YZ-TCRO-ZY where: Y=acation Z=a polar group which is associated with the cation, andTCRO=trans carotenoid skeleton comprising the steps of: a) preparingdeionized water, b) adding a BTCS to the solution of step a).
 13. Amethod as in claim 9, 10, 11 or 12 wherein said compound is trans sodiumcrocetinate.
 14. A method of increasing the diffusivity of oxygen in amammal comprising administering to a mammal a therapeutically effectiveamount of a compound having the formula: YZ-TCRO-ZY where: Y=a cationZ=a polar group which is associated with the cation, and TCRO=transcarotenoid skeleton, wherein said compound is not TSC.
 15. A method asin claim 14 wherein said administration is by inhalation.
 16. A methodof treating respiratory disease comprising administering to a mammal inneed of treatment a therapeutically effective amount of a compoundhaving the formula: YZ-TCRO-ZY where: Y=a cation Z=a polar group whichis associated with the cation, and TCRO=trans carotenoid skeleton,wherein said compound is not TSC.
 17. A method of treating emphysemacomprising administering to a mammal in need of treatment atherapeutically effective amount of a compound having the formulaYZ-TCRO-ZY where: Y=a cation Z=a polar group which is associated withthe cation, and TCRO=trans carotenoid skeleton, wherein said compound isnot TSC.
 18. A method of treating hemorrhagic shock comprisingadministering to a mammal in need of treatment a therapeuticallyeffective amount of a compound having the formula YZ-TCRO-ZY where: Y=acation Z=a polar group which is associated with the cation, andTCRO=trans carotenoid skeleton, wherein said compound is not TSC.
 19. Amethod of treating cardiovascular disease comprising administering to amammal in need of treatment a therapeutically effective amount of acompound having the formula YZ-TCRO-ZY where: Y=a cation Z=a polar groupwhich is associated with the cation, and TCRO=trans carotenoid skeleton,wherein said compound is not TSC.
 20. A method of treatingatherosclerosis comprising administering to a mammal in need oftreatment a therapeutically effective amount of a compound having theformula YZ-TCRO-ZY where: Y=a cation Z=a polar group which is associatedwith the cation, and TCRO=trans carotenoid skeleton, wherein saidcompound is not TSC.
 21. A method of treating asthma comprisingadministering to a mammal in need of treatment a therapeuticallyeffective amount of a compound having the formula YZ-TCRO-ZY where: Y=acation Z=a polar group which is associated with the cation, andTCRO=trans carotenoid skeleton, wherein said compound is not TSC.
 22. Amethod of treating spinal cord injuries comprising administering to amammal in need of treatment a therapeutically effective amount of acompound having the formula YZ-TCRO-ZY where: Y=a cation Z=a polar groupwhich is associated with the cation, and TCRO=trans carotenoid skeleton,wherein said compound is not TSC.
 23. A method of treating cerebraledema comprising administering to a mammal in need of treatment atherapeutically effective amount of a compound having the formulaYZ-TCRO-ZY where: Y=a cation Z=a polar group which is associated withthe cation, and TCRO=trans carotenoid skeleton, wherein said compound isnot TSC.
 24. A method of treating papillomas comprising administering toa mammal in need of treatment a therapeutically effective amount of acompound having the formula YZ-TCRO-ZY where: Y=a cation Z=a polar groupwhich is associated with the cation, and TCRO=trans carotenoid skeleton,wherein said compound is not TSC.
 25. A method of treating hypoxiacomprising administering to a mammal in need of treatment atherapeutically effective amount of a compound having the formulaYZ-TCRO-ZY where: Y=a cation Z=a polar group which is associated withthe cation, and TCRO=trans carotenoid skeleton, wherein said compound isnot TSC.
 26. A method of synthesizing a BTCS compound having the formulaYZ-TCRO-ZY where: Y=a cation Z=a polar group which is associated withthe cation, and TCRO=trans carotenoid skeleton, comprising the steps of:a) coupling a symmetrical dialdehyde containing conjugated carbon-carbondouble bonds with a triphenylphosphorane, b) saponifying the product ofstep a).
 27. A method as in claim 26 wherein the coupling of step a) ismade using [3-carbomethoxy-2-buten-1-ylidene]triphenylphosphorane.
 28. Amethod as in claim 26 wherein the product of step a) is saponified usinga solution of NaOH and methanol.
 29. A method as in claim 26 whereinafter step a) is the step of isolating the desired product of thecoupling reaction.
 30. A method of saponifying a symmetrical diestercontaining conjugated carbon-carbon double bonds to form a BTCS,comprising the steps of: a) solubilizing the symmetrical diestercontaining conjugated carbon-carbon double bonds with a compoundselected from the group consisting of methanol, ethanol, propanol andisopropanol, and b) mixing the solution of step a) with a base.
 31. Amethod as in claim 30 wherein the base is selected from the groupconsisting of NaOH, KOH, and LiOH.
 32. A method as in claim 30 whereinthe diester is saponified using methanol and NaOH.
 33. A BTCS compoundsynthesized according to claim
 26. 34. A BTCS composition whereinabsorbency of the highest peak which occurs in the visible wave lengthrange divided by the absorbency of the peak which occurs in the UV wavelength range is greater than 7.5.
 35. A TSC composition whereinabsorbency of the highest peak which occurs in the visible wave lengthrange divided by the absorbency of the peak which occurs in the UV wavelength range is greater than 7.5.
 36. A method of increasing thediffusivity of oxygen in a mammal comprising administering to a mammal atherapeutically effective amount of BTCS wherein absorbency of thehighest peak which occurs in the visible wave length range divided bythe absorbency of the peak which occurs in the UV wave length range isgreater than 7.5.
 37. A method of treating emphysema comprisingadministering to a mammal in need of treatment a therapeuticallyeffective amount of BTCS wherein absorbency of the highest peak whichoccurs in the visible wave length range divided by the absorbency of thepeak which occurs in the UV wave length range is greater than 7.5.
 38. Amethod of treating hemorrhagic shock comprising administering to amammal in need of treatment a therapeutically effective amount of BTCSwherein absorbency of the highest peak which occurs in the visible wavelength range divided by the absorbency of the peak which occurs in theUV wave length range is greater than 7.5.
 39. A method as in claim 36,37 or 38 wherein the BTCS is TSC.
 40. A method of increasing thediffusivity of oxygen in a mammal comprising administering to a mammalby inhalation a therapeutically effective amount of TSC.
 41. An inhalercontaining a BTCS compound having the structure: YZ-TCRO-ZY where: Y=acation Z=polar group which is associated with the cation, and TCRO=transcarotenoid skeleton.
 42. An inhaler as in claim 40 wherein said BTCScompound is TSC.
 43. A method of converting an isomeric mixture ofolefinic dialdehydes into the all trans aldehyde comprising isomerizingsaid isomeric mixture of dialdehydes with a sulfinic acid in a solvent.44. A method as in claim 43 wherein said sulfinic acid has the formulaRSO₂H where R is C1 through C10 straight or branched alkyl group or anaryl group.
 45. A method as in claim 43 where the solvent is selectedfrom the group consisting of 1,4-dioxane, tetrahydrofuran or dialkylether wherein the alkyl group is a C1 through C10 straight or branchedalkyl group.
 46. A method as in claim 43 wherein said sulfinic acid ispara-toluenesulfinic acid and said solvent is 1,4-dioxane.
 47. A methodas in claim 43 wherein said olefinic dialdehyde is2,7-dimethyl-2,4,6-ocatrienedial.
 48. A method as in claim 43 whereinsaid olefinic dialdehyde is 2,7-dimethyl-2,4,6-ocatrienedial, saidsulfinic acid is para-toluenesulfinic acid and said solvent is1,4-dioxane.
 49. A method of treating ischemia comprising administeringto a mammal in need of treatment a therapeutically effective amount of acompound having the formula: YZ-TCRO-ZY where: Y=a cation Z=a polargroup which is associated with the cation, and TCRO=trans carotenoidskeleton, wherein said compound is not TSC.
 50. A method of treatingtraumatic brain injury comprising administering to a mammal in need oftreatment a therapeutically effective amount of a compound having theformula YZ-TCRO-ZY where: Y=a cation Z=a polar group which is associatedwith the cation, and TCRO=trans carotenoid skeleton, wherein saidcompound is not TSC.
 51. A method of enhancing performance of a mammalcomprising administering to said mammal a therapeutically effectiveamount of a compound having the formula: YZ-TCRO-ZY where: Y=a cationZ=a polar group which is associated with the cation, and TCRO=transcarotenoid skeleton.
 52. A method of treating complications of diabetescomprising administering to a mammal in need of treatment atherapeutically effective amount of a compound having the formulaYZ-TCRO-ZY where: Y=a cation Z=a polar group which is associated withthe cation, and TCRO=trans carotenoid skeleton.
 53. A method of treatingAlzheimer's disease comprising administering to a mammal in need oftreatment a therapeutically effective amount of a compound having theformula YZ-TCRO-ZY where: Y=a cation Z=a polar group which is associatedwith the cation, and TCRO=trans carotenoid skeleton.
 54. A method oftreating ischemia in a mammal comprising administering to a mammal atherapeutically effective amount of BTCS wherein absorbency of thehighest peak which occurs in the visible wave length range divided bythe absorbency of the peak which occurs in the UV wave length range isgreater than 7.5.
 55. A method of treating traumatic brain injurycomprising administering to a mammal in need of treatment atherapeutically effective amount of BTCS wherein absorbency of thehighest peak which occurs in the visible wave length range divided bythe absorbency of the peak which occurs in the UV wave length range isgreater than 7.5.
 56. A method of enhancing performance comprisingadministering to a mammal an effective amount of BTCS wherein absorbencyof the highest peak which occurs in the visible wave length rangedivided by the absorbency of the peak which occurs in the UV wave lengthrange is greater than 7.5.
 57. A method of treating diabetes comprisingadministering to a mammal in need of treatment a therapeuticallyeffective amount of BTCS wherein absorbency of the highest peak whichoccurs in the visible wave length range divided by the absorbency of thepeak which occurs in the UV wave length range is greater than 7.5.
 58. Amethod of treating Alzheimer's disease comprising administering to amammal in need of treatment a therapeutically effective amount of BTCSwherein absorbency of the highest peak which occurs in the visible wavelength range divided by the absorbency of the peak which occurs in theUV wave length range is greater than 7.5.
 59. A method as in claim 54,55, 56, 57 or 58 wherein the BTCS is TSC.
 60. A method of treating,preventing or reducing the amount of ischemia resulting from surgery ofa mammal comprising administering to a mammal before, during or aftersurgery a therapeutically effective amount of BTCS.